High density peptide polymers

ABSTRACT

Described herein, inter alia, are compositions of cell penetrating high density brush peptide polymer, drug delivery vehicles for delivering cell penetrating peptides and drugs. Also provided is a methodology for protecting active peptides from proteolysis by packaging them into high-density brush polymers via ring opening metathesis polymerization (ROMP), using an easily-prepared catalyst initiator. The graft-through polymerization of norbomyl-peptide monomers via ROMP can result in structures that resist proteolysis relative to their monomeric analogues in a manner dependent on their degree of polymerization (REFS). Polymerized peptides, while protected from proteolysis, maintain their intended biological function and are capable of any of the functions inherent to the peptide, such as binding a receptor or ligand, initiating a signaling pathway, penetrating a cell, or inducing a therapeutic effect.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/035,313, filed Aug. 8, 2014; which is incorporatedherein by reference in its entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under 1DP2OD008724,1R01EB011633, and R01HL117326, awarded by the National Institutes ofHealth. The government has certain rights in this invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII FILE

The Sequence Listing written in file 48537-560001WO_ST25.TXT, created onAug. 9, 2015, 25,739 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND

Peptides have been developed for decades for use as therapeutics,signaling agents, and sensors. However, significant problems limit theiruse in vivo, notably the short duration of their activity, due toexpeditious digestion by endogenous proteases and efficient renalclearance due to their generally low molecular weights. Proteolyticdigestion of circulating peptides can be rapid, occurring withhalf-lives of less than a few minutes, owing to the abundance of activeproteases in both serum and tissues. The greatest threats to peptideintegrity are found in the lumen of the small intestine, which containsgram quantities of proteases secreted by the pancreas (i.e.α-chymotrypsin, trypsin, and carboxypeptidases), as well as in the brushborder membrane of epithelial cells, which houses some 15 peptidasesthat together cleave amide bonds in peptides and proteins with littlespecificity. In practice, unmodified therapeutic peptides are typicallydirectly injected at the site of interest to minimize proteolyticdegradation, and many are used only as last-resort, salvage treatmentsin patients with multi-drug resistant afflictions. Harnessing theinherent specificity, affinity, and low immunogenicity of peptides intherapeutic and diagnostic applications will require the development ofsimple, widely applicable, and easy-to-access methods that protectactive peptides from proteolysis, but do not hinder their function.

Traditional strategies for limiting enzymatic degradation typicallyinvolve chemical modification of the peptide, such as by incorporatingunnatural amino acids (including the use of D-amino acids), terminalcapping via acetylation of the N-terminus or amidation of theC-terminus, introduction of backbone modifications such asN-methylation, use of stabilizing linkers, and conjugation topolyethylene glycol (PEG). Hence, chemistries are chosen such thatpeptides are no longer recognized by, or become inaccessible to, theactive site of a proteolytic enzyme. However, because these strategiesmodify the connectivity or amino acid identity of the peptide, eachmodification has the potential to impact the ability of the peptide toelicit its intended response, often necessitating multiple rounds ofstructure-function studies to verify the activity of the material.Strategies that do not require direct modification of the peptidechemical structure typically involve manipulation of theirthree-dimensional spatial arrangement via chemical conjugation of thepeptide to a higher molecular weight structure. Architectures of thistype include polymeric, or nanoparticle conjugates or systems involvingthe display of multiple copies of the peptide on a small moleculescaffold. However, in practice syntheses of these materials oftenrequire multiple conjugation and purification steps or the preparationof complicated scaffolds that are not generalizable or convenient.

In addition to proteolysis, there are two other problems that oftenlimit the bioavailability and clinical efficacy of peptide-basedtherapeutics: rapid renal clearance and inefficiencies in cellularuptake. Regarding the former, it should be noted that the exclusionlimit for glomerular filtration includes molecules or assemblies whosemolecular weight exceeds 50 kDa.

Provided herein are solutions to these and other problems.

BRIEF SUMMARY OF THE DISCLOSURE

The disclosure herein describes, inter alia, a methodology forprotecting active peptides from proteolysis by packaging them intohigh-density brush polymers via ring opening metathesis polymerization(ROMP), using an easily-prepared catalyst initiator. The graft-throughpolymerization of norbornyl-peptide monomers via ROMP can result instructures that resist proteolysis relative to their monomeric analoguesin a manner dependent on their degree of polymerization (REFS).Polymerized peptides, while protected from proteolysis, maintain theirintended biological function and this phenomenon is a general feature ofpeptides arranged in this manner. Such an approach provides a general,accessible route to the development of proteolytically-resistant peptidedisplays, capable of any of the functions inherent to the peptide, suchas binding a receptor or ligand, initiating a signaling pathway,penetrating a cell, or inducing a therapeutic effect. Thus, the currentdisclosure provides, inter alia, compositions of cell penetrating highdensity brush peptide polymer, drug delivery vehicles for deliveringcell penetrating peptides and drugs, and methods of preparing the same.

In an aspect is provided a polymer having the formula: R¹-[M(O)]_(n)—R²wherein, n is an integer from 2 to 1000; M is a polymerized monomer; Ois independently a polypeptide (a polypeptide moiety) covalentlyattached to M through a covalent linker; and R¹ and R² are independentlyterminal polymer moieties.

In an aspect is provided a block copolymer having the formula:R¹-[M(O)]_(n)-[M(P)]_(m)—R² or R¹-[M(P)]_(m)-[M(O)]_(n)—R² wherein, n isan integer from 2 to 1000; m is an integer from 2 to 1000; M is apolymerized monomer; O is independently a polypeptide (a polypeptidemoiety) covalently attached to M through a covalent linker; P isindependently a non-polypeptide moiety; and R¹ and R² are independentlyterminal polymer moieties.

In an aspect is provided a blend copolymer having the formula:R¹-([M(O)]_(n)-[M(P)]_(m))_(z)—R² or R¹-([M(P)]_(m)-[M(O)]_(n))_(z)—R²wherein, n is an integer from 2 to 1000; m is an integer from 2 to 1000;M is a polymerized monomer; O is independently a polypeptide (apolypeptide moiety) covalently attached to M through a covalent linker;P is independently a non-polypeptide moiety; z is an integer from 2 to100; and R¹ and R² are independently terminal polymer moieties.

In an aspect is provided a micelle including a polymer (e.g., copolymer)described herein.

In an aspect is provided a nanoparticle including a polymer (e.g.,copolymer) described herein.

In another aspect is provided a pharmaceutical composition including apharmaceutically acceptable excipient and a polymer (e.g., polypeptidepolymer, copolymer, block copolymer, blend copolymer), micelle, ornanoparticle (e.g, each as described herein).

In an aspect is provided a method of administering a polypeptide to theinterior of a cell including contacting the cell with a polymer (e.g.,polypeptide polymer, copolymer, block copolymer, blend copolymer),micelle, or nanoparticle (e.g, each as described herein).

In an aspect is provided a method of internalizing polypeptides (e.g.,polypeptides copolymers, block copolymers, blend copolymers) into a cellincluding contacting the cell with a polymer.

In an aspect is provided a method of treating a disease in a subject,including administering to the subject an effective amount of a polymerdescribed herein (e.g., polypeptide polymer, copolymer, block copolymer,blend copolymer), micelle, or nanoparticle (e.g., each as describedherein).

In one aspect, the present disclosure provides a composition including acell penetrating high density brush peptide polymer. The peptide polymermay include a polypeptide (i.e. polypeptide moiety) with arginine orlysine residues appended at the N- or at the C-terminus, and thepolypeptide is pendant to polymerized norbornene moieties (thepolymerized monomer).

In one aspect, the present disclosure provides a drug delivery vehiclefor delivering a therapeutic agent into a cell, including a cellpenetrating high density brush peptide polymer, where the peptidepolymer includes polypeptide (polypeptide moiety) with arginine orlysine residues appended at the N- or at the C-terminus, and polypeptideis pendant to polymerized norbornene moieties (the polymerized monomer).

In another aspect, the present disclosure provides a method of preparinga cell penetrating high density brush peptide polymer, where the peptidepolymer includes a polypeptide (polypeptide moiety) with an arginine ora lysine residue appended to the N- or at the C-terminus, and is pendantto a polymerized norbornene moieties (the polymerized monomer). Themethod involves combining a plurality of polypeptide monomers (i.e. anunpolymerized monomer attached to a polypetide) with a rutheniumcatalyst into a mixture for ring opening metathesis polymerization(ROMP).

In another aspect, the present disclosure provides a method ofdelivering a peptide, a pharmaceutically active agent or anoligonucleotide to a cell. The method includes contacting a cell with apolymer provided herein. The method may employ a delivery devicedisclosed herein.

Other features and advantages of the disclosure will be apparent fromthe following detailed description and claims.

Unless noted to the contrary, all publications, references, patentsand/or patent applications reference herein are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic showing synthetic routes for the polymerizationof cell penetrating polymers and controls, and routes to the preparationof homopolymers. Sequence legend: GSGSG (SEQ ID NO: 1); YGRKKRRQRRR (SEQID NO:2); RRRRRRRR (SEQ ID NO:3).

FIG. 1B is a schematic showing synthetic routes for the polymerizationof cell penetrating polymers and controls, and routes to the preparationof block copolymers. Note that the guanidinium moiety and Arg8 peptideare polymerized with protecting groups and deprotected afterpolymerization by treatment of the polymers with a TFA solution. Foreach polymer, m and n are the degrees of polymerization (DPs).

FIG. 2A is a TEM (transmission electron microscopy) image for the Tatparticle. Scale bar is 200 nm

FIG. 2B is a TEM image for the GSGSG particle. Scale bar is 200 nm.

FIG. 3 is a bar graph showing the quantitative comparison of cellularuptake of peptides, polymers and particles at 2.5 μM after 30 minincubation with HeLa cells by flow cytometry. On the y-axis, normalizedmean fluorescence refers to the mean fluorescence counts detected forthe material divided by the mean fluorescence counts exhibited by thevehicle control (PBS).

FIG. 4 are images showing live-cell confocal images of peptides,polymers and nanoparticles labeled with fluorescein. Images are theaverage maximum intensity from six consecutive 1 μm slices. Scale barsare 50 m.

FIG. 5A is a histogram bar graph showing the pharmacological and thermalprobes of cellular uptake mechanisms. HeLa cells were pretreated withMβCD (9.5 mM) or dynasore (80 μM) for 30 min prior to incubation withthe material of interest or preincubated at 4° C. For each bin of thehistogram, treatments were (left to right): No treatment, MβCD (5 mM),Dynasore (80 μM), and 4° C.

FIG. 5B is a histogram bar graph showing the concentration dependence ofthe cellular uptake of key materials. All reported flow cytometry dataare described as a fold-shift relative to the vehicle control. Allexperiments described here were performed in DMEM with 10% FBS. For eachbin of the histogram, concentrations (μM) were (left to right): 12.5,2.5, 1.25, 0.5, and 0.25.

FIG. 6A is a histogram bar graph showing RP-HPLC (reverse-phase highpressure liquid chromatography) of the quantity of remaining materialpostenzymatic treatment. For each bin of the histogram, proteincomponent was (left to right): trypsin, chymotrypsin, and pronase.

FIG. 6B is a histogram bar graph showing flow cytometry data of thematerials after proteolytic digestion. Data is reported as thepercentage of fluorescence seen after enzyme treatment relative to thevalue seen without treatment. For each bin of the histogram,concentrations (μM) were (left to right): 12.5, 2.5, 1.25, 0.5, and0.25.

FIG. 6C depict confocal microscopy images comparing cells incubated withmaterials that have been pretreated with chymotrypsin alongside cellsincubated with materials that have not received this pretreatment.Images are the maximum average intensity from six consecutive 1 μmslices. Scale bars are 50 m. In all cases Tat-containing materials atthe indicated concentrations were treated with 1 μM of protease for 20min at 37° C. Left column: no protease; right column: after protease.

FIG. 7A is the chemical structure of a set of homopolymers containing athrombin peptide substrate. Sequence legend: GALVPRGSGERDG (SEQ IDNO:4).

FIG. 7B is a graph showing the cleavage kinetics of thrombin-sensitivemonomers and homopolymers at DP (degree of polymerization)=10, 20 and30.

FIG. 7C is a chemical structure of the fluorogenic substratehomopolymer. Sequence legend: E(EDANS)RPAHLRDSGK(DABCYL)GSGSG (SEQ IDNO:5).

FIG. 7D is a graph showing the cleavage kinetics of the fluorogenichomopolymer relative to the monomer, by multiple proteases in additionto the protease for which the substrate is optimized, MT1-MMP (matrixmetalloprotease).

FIG. 8A is a chemical structure of a series of random blend copolymersof the fluorogenic peptide substrate monomer and an OEG (oligoethyleneglycol) monomer.

FIG. 8B is a graph showing the comparison of the cleavage kinetics ofthe fluorogenic random blend copolymers, (ratio is m:n as shown in A,overall DP=20) homopolymer (DP=20) and monomer.

FIG. 9A is a chemical structure of the homopolymer with ten fluorogenicpeptides; Monomer=NorG-E(EDANS)RPAHLRDSGK(DABCYL)GSGSG), MW=23.2 kDa;N=200.

FIG. 9B is a representative conformation and surface-accessibility datafor in silico models of the homopolymer.

FIG. 9C is a chemical structure of the 9:1 ratio blend copolymer withthe peptide at the end of the polymer where nine of the norbornenemoieties are linked to OEG-4 and the tenth, at position 10 of thenorbornene chain, is linked to the fluorogenic peptide, MW=5.6 kDa,N=65. Simulated polymer without dye components (same chemicalstructure); MW=5.11 kDa, N=63.

FIG. 9D is a representative conformation and surface-accessibility datafor in silico models of the peptide at the end of the polymer.

FIG. 9E is a chemical structure of the 9:1 ratio blend with the peptidein the middle (i.e., position 5), blend polymer where the peptide islinked to the fifth norbornene of the chain, MW=5.6 kDa, N=65. Simulatedpolymer without dye components (same chemical structure); MW=5.11 kDa,N=63.

FIG. 9F is a representative conformation and surface-accessibility datafor in silico models of the peptide at the end of the polymer.

FIG. 9G is a chemical structure of the intermediate 5:5 ratio blendcopolymer, where five norbornene moieties are linked to the fluorogenicpeptide and the other five are linked to OEG-4; MW=13.4 kDa, N=125.

FIG. 9H is a representative conformation and surface-accessibility datafor in silico models of the intermediate 5:5 ratio blend copolymer.

FIG. 9I is a plot showing the probe-accessible surface area of the fourstructures (11A-11H), averaged over the last 40 ns of each heat-coolcycle. Blue bars represent the surface area accessible per peptide to aspherical probe with a radius of 3.14 nm (size on the order of a typicalprotease) and the gray bars represent the same measurement using a proberadius of 0.14 nm (approximately the size of a water molecule).

FIG. 10 are chemical structures of monomers used in cell penetrationstudies. The side-chains on the arg8 monomer are protected with a Pbf(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl chloride) group.

FIG. 11 is a bar graph showing cell cytotoxicity studies using theCellTiter Blue Assay. Percentages are relative to vehicle control. Cellswere treated for 48 hours with the Tat and GSGSG peptide, polymer andparticle at 5 μM. The positive control is 10% DMSO. Cells treated withany peptide-based material remained at least 92% viable.

FIG. 12 is a chemical structure of thrombin substrate monomer.

FIG. 13 is a chromatogram showing proteolytic digestion of the monomerat t=12 hours. The chromatogram of the polymer at t=0 is purple and thereaction at t=12 is black with gradient at 0-80% Buffer B.

FIG. 14 are chemical structures of fluorogenic monomers prepared.Sequence legend:

(SEQ ID NO: 5) E(EDANS)RPAHLRDSGK(DABCYL)GSGSG; (SEQ ID NO: 7)K(DABCYL)RPAHLRDSGE(EDANS)GSGSG; (SEQ ID NO: 9))GSGSGE(EDANS)RPAHLRDSGK(DABCYL).

FIG. 15A is a schematic showing polymerization of fluorogenic monomerinto a homopolymer.

FIG. 15B is a schematic showing polymerization of fluorogenic monomerinto a random blend co-polymer with an OEG monomer.

FIG. 16 is a schematic showing the fluorescent assay used to detectcleavage of the fluorogenic substrate by MT1-MMP. In the intact monomer(NorGly-E(EDANS)-RPAHLRDSGK(dabcyl)GSGSG-NH₂) (SEQ ID NO: 11), thefluorescence of the donor, EDANS is quenched by the acceptor, DABCYLuntil cleavage of the substrate by the protease. Note that the otherproteases used in this study will cleave at other locations along thepeptide substrate, but scission at any amide bond of the substratesequence will result in liberation of the quencher and the onset offluorescence.

FIG. 17 is a graph showing the time course of the proteolysis of thefluorogenic monomer and homopolymer by MT1-MMP and MMP-9 (for which theamino acid sequence should not be a substrate). While the monomer iscleaved readily by MT1-MMP, MMP-9 shows little activity. Neither enzymewas able to proteolytically digest the homopolymer.

FIG. 18 is a graph showing the time course of proteolysis of fluorogenichomopolymers and their monomer building blocks (40 μM) by MT1-MMP (25nM). Chemical structures are shown in FIG. 14. Little proteolysis of thehomopolymers was observed regardless of the location of the quencher ordonor or whether a five-amino acid linker (GSGSG) was used to space thesubstrate from the polymer backbone.

FIG. 19A is a chemical structure showing the peptide block copolymers.Sequence legend: GSGSG (SEQ ID NO:1); RGSGSG (SEQ ID NO:12); RRGSGSG(SEQ ID NO:13); GSGSGR (SEQ ID NO:14); GSGSGRR (SEQ ID NO:15); GSGSGK(SEQ ID NO:16); GSGSGKK (SEQ ID NO:17).

FIG. 19B is a bar graph showing flow cytometry data of fluorescentsignatures of HeLa cells treated with the polymers (m˜60) and theirmonomeric counterparts. All data are normalized to the vehicle (DPBS),which is assigned a value of 1. The R control is a block copolymer thatcontains a single Arg attached via a short linker at each polymer sidechain of the first block (m˜60). “Flu” is the fluorescein end-labelshown in FIG. 19A

FIG. 20A is a chemical structure showing the homopolymers. “Flu” is thefluorescein-end label. Sequence legend: KLAKLAKKLAKLAK (SEQ ID NO: 18).

FIG. 20B is a bar graph showing flow cytometry data showing fluorescentsignatures of HeLa cells treated with the KLA polymers and peptide. Datais normalized to DPBS at a value of 1.

FIG. 20C is a graph showing Viability of cells treated with KLA polymers(m ˜5), GSGSGRR (m˜60) polymer, GSGSGKK (m ˜60) polymer and the KLApeptide. LD₅₀ values for the KLA polymers, obtained by fitting data tothe Hill equation, are 12.5, 25, and 30 μM for the m˜5, 10 and 15polymers, respectively. Note that the dose-response curves for the m˜10and 15 KLA polymers are provided in FIG. 29.

FIG. 20D is a graph showing mitochondrial membrane potential disruptionassays. The percentages given describe the percent of signal for eachmaterial in the disrupted mitochondria region.

FIG. 21A is a histogram bar graph showing flow cytometry data describingpharmacological inhibition of dynamin-mediated endocytosis by dynasore,membrane fluidity by methyl-β-cyclodextrin (M-βCD) or membranetrafficking by a reduction in incubation temperature. Data is normalizedto DPBS at a value of 1. Each bin of the histogram (left to right):untreated, MBCD, Dynbasore, 4° C.

FIG. 21B is a bar graph showing RP-HPLC chromatograms of the materialbefore and after treatment with trypsin or the protease cocktail Pronasethat was used to determine proteolytic susceptibility.

FIG. 22 is a chemical structure showing ROMP monomers. The GSGSGKKpeptide and all KLA variants were prepared without protecting groups forenhanced solubility in DMF in preparation for polymerization. Sequencelegend: GSGSG (SEQ ID NO: 1); R(Pbf)GS(OtBu)GS(OtBu)G (SEQ ID NO: 19);R(Pbf)R(Pbf)GS(OtBu)GS(OtBu)G (SEQ ID NO:20); GS(OtBu)GS(OtBu)GR(Pbf)(SEQ ID NO:21); GS(OtBu)GS(OtBu)GR(Pbf)R(Pbf) (SEQ ID NO:22);GS(OtBu)GS(OtBu)GK(Boc) (SEQ ID NO:23); GSGSGKK (SEQ ID NO:17);KLAKLAKKLAKLAK (SEQ ID NO:18); KLAKLAK (SEQ ID NO:24).

FIG. 23 is a schematic showing the polymerization scheme for thepreparation of GSGSG peptide polymers and analogues. Peptide polymersfor this series are prepared via ROMP as block copolymers with anaccessory OEG block to ensure aqueous solubility of all GSGSG analogues.Each peptide block is polymerized to m˜8, 15, 30, and 60. The OEG blockis kept constant at a DP of ˜20. The living polymer can be terminatedwith ethyl vinyl ether after completion of the peptide block to gainaccurate molecular weights (M_(n)), dispersity (M_(w)/M_(n)) and degreeof polymerization (DP). To monitor uptake of the materials, each polymeris end-labeled with fluorescein. Polymers used in all toxicity studies,were prepared without a fluorophore by terminating the polymerizationwith ethyl vinyl ether prior after addition of the second (OEG) block.

FIG. 24 is a schematic showing the polymerization scheme for thepreparation of KLA homopolymers. KLA peptide polymers were prepared ashomopolymers (m˜5, 10, 15) via ROMP and end-labeled with fluorescein aswith the block copolymers. Identical conditions were used to prepare theKLAKLAK (fragment) control polymer. Polymers used in all toxicitystudies, were prepared without a fluorophore by terminating thepolymerization with ethyl vinyl ether after the peptide has finishedpolymerizing. Sequence legend: KLAKLAKKLAKLAK (SEQ ID NO: 18).

FIG. 25 is a histogram bar graph showing the influence of the degree ofpolymerization or “m” on cellular uptake as quantified by flowcytometry. All data are referenced to vehicle (DPBS), which gives avalue of 1. The concentration for each material is 2.5 μM, whereconcentration is with respect to the fluorophore. For each histogram bin(left to right); 8-mer, 14-mer, 30-mer and 60-mer.

FIG. 26 is a histogram bar graph showing the concentration dependence ofcellular uptake as quantified by flow cytometry. All data are referencedto vehicle (DPBS), which gives a value of 1. Concentration of allmaterials is with respect to the fluorophore. For each histogram bin(left to right): 2.5 μM, 5 μM, 12.5 μM.

FIG. 27 are live cell confocal microscopy images of peptides andpolymers. All images are the average intensity from six consecutive 1 μmZ-slices using a 40× objective. Cells were treated with 2.5 μM of thematerial (with respect to fluorophore). All GSGSG polymers and analogueshave a peptide m of ˜60 and the KLA polymer is m˜10. Scale bars are 50μm. Each polymer that contains Lys or Arg shows a mixture of diffuse andpunctate fluorescence, indicating that the material resides in thecytosol and in cellular compartments, respectively.

FIG. 28 are live-cell confocal microscopy images of R control (DP or mof ˜60), KLAfragment (m ˜10) and Tat peptide. All images are the averageintensity from six consecutive 1 μm Z-slices using a 40× objective.Scale bars are 50 μm. Each image shows a mixture of diffuse and punctatefluorescence, indicating that the material resides in the cytosol and incellular compartments, respectively.

FIG. 29 is a graph showing a comparison of cytotoxicity curves for KLAmaterials and controls. This figure is identical to that of FIG. 20C,except it also contains the dose-response curves for the KLA (fulllength) polymers at m˜10 and 15.

FIG. 30A is a spectra showing UV-Vis circular dichroism of the KLApeptide. Since spectra for the full length and polymer are nearlyidentical, polymerization of the KLA sequence does not perturb thesecondary structure of the peptide.

FIG. 30B is a spectra showing UV-Vis circular dichroism of the KLAhomopolymer polymer (m˜10).

FIG. 30C is a spectra showing UV-Vis circular dichroism of the KLAfragment polymer with m˜10. The spectra are different from the fulllength constructs, indicating that the secondary structure for thismaterial is unique.

FIG. 30D is a spectra of an overlay of FIGS. 30A (KLA peptide), 30B (KLAhomopolymer m˜10) and 30C (KLA fragment polymer m˜10).

FIG. 31A is a graph showing flow cytometry data for the vehicle control(DPBS) in a JC-1 (mitochondrial dye) assay for mitochondrial integrity.The quadrants were chosen based on where cells treated with CCCP (Q3)and cells that were untreated reside (Q2), with the line drawn betweenthese two regions. Q2 is the percent of cells with healthy mitochondria,and Q3 is the percent of cells with the unhealthy mitochondria.

FIG. 31B is a graph showing flow cytometry data for JC-1 only. Thequadrants were chosen based on where cells treated with CCCP (Q3) andcells that were untreated reside (Q2), with the line drawn between thesetwo regions. Q2 is the percent of cells with healthy mitochondria, andQ3 is the percent of cells with the unhealthy mitochondria.

FIG. 31C is a graph showing flow cytometry data for the JC-1+CCCP(Carbonyl cyanide m-chlorophenyl hydrazone) in a JC-1 (mitochondrialdye) assay for mitochondrial integrity. The quadrants were chosen basedon where cells treated with CCCP (Q3) and cells that were untreatedreside (Q2), with the line drawn between these two regions. Q2 is thepercent of cells with healthy mitochondria, and Q3 is the percent ofcells with the unhealthy mitochondria.

FIG. 31D is a graph showing flow cytometry data for KLA (m˜5) in a JC-1(mitochondrial dye) assay for mitochondrial integrity. The quadrantswere chosen based on where cells treated with CCCP (Q3) and cells thatwere untreated reside (Q2), with the line drawn between these tworegions. Q2 is the percent of cells with healthy mitochondria, and Q3 isthe percent of cells with the unhealthy mitochondria.

FIG. 31E is a graph showing flow cytometry data for KLA (m˜10) in a JC-1(mitochondrial dye) assay for mitochondrial integrity. The quadrantswere chosen based on where cells treated with CCCP (Q3) and cells thatwere untreated reside (Q2), with the line drawn between these tworegions. Q2 is the percent of cells with healthy mitochondria, and Q3 isthe percent of cells with the unhealthy mitochondria.

FIG. 31F is a graph showing flow cytometry data for KLA (m˜15) in a JC-1(mitochondrial dye) assay for mitochondrial integrity. The quadrantswere chosen based on where cells treated with CCCP (Q3) and cells thatwere untreated reside (Q2), with the line drawn between these tworegions. Q2 is the percent of cells with healthy mitochondria, and Q3 isthe percent of cells with the unhealthy mitochondria.

FIG. 31G is a graph showing flow cytometry data for KLA fragment (m˜10)in a JC-1 (mitochondrial dye) assay for mitochondrial integrity. Thequadrants were chosen based on where cells treated with CCCP (Q3) andcells that were untreated reside (Q2), with the line drawn between thesetwo regions. Q2 is the percent of cells with healthy mitochondria, andQ3 is the percent of cells with the unhealthy mitochondria.

FIG. 31H is a graph showing flow cytometry data for GSGSGRR (SEQ ID NO:15) (m˜60) in a JC-1 (mitochondrial dye) assay for mitochondrialintegrity. The quadrants were chosen based on where cells treated withCCCP (Q3) and cells that were untreated reside (Q2), with the line drawnbetween these two regions. Q2 is the percent of cells with healthymitochondria, and Q3 is the percent of cells with the unhealthymitochondria.

FIG. 31I is a graph showing flow cytometry data for GSGSGKK (SEQ ID NO:17) (m˜60) in a JC-1 (mitochondrial dye) assay for mitochondrialintegrity. The quadrants were chosen based on where cells treated withCCCP (Q3) and cells that were untreated reside (Q2), with the line drawnbetween these two regions. Q2 is the percent of cells with healthymitochondria, and Q3 is the percent of cells with the unhealthymitochondria.

FIG. 32 is a bar graph showing the levels of Caspase 3/7 in untreatedcells and cells treated with KLA polymers at varying DP. The baselinelevel of expression is taken as 100% in the untreated cells. Thosetreated with polymers show an increase in expression, which isindicative of apoptosis. Each measurement was performed 3-fold on atleast two separate subcultures.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein, inter alia, are cell penetrating high density brushpeptide polymer compositions and/or delivery vehicles for deliveringcell penetrating peptides and drugs, and methods of preparing the same.

Definitions

The following definitions are included for the purpose of understandingthe present subject matter and for constructing the appended patentclaims. Abbreviations used herein have their conventional meaning withinthe chemical and biological arts.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this disclosure. The followingdefinitions are provided to facilitate understanding of certain termsused frequently herein and are not meant to limit the scope of thepresent disclosure.

The term “polypeptide polymer” refers to a polymer of polypeptides thatcontains a polymer backbone with a polypeptide attached to a polymerizedmonomer at multiple positions (e.g., every position, periodic position)of the backbone (i.e., a polypeptide connected at one end of thepolypeptide to the polymer monomer moiety). In embodiments, thepolypeptide polymer is a polymer of norbornyl monomer (the polymerizedmonomer) attached to apolypeptide (e.g., 2-2000 amino acids) (alsoreferred to herein as a polypeptide moiety) forming a brush polymerwhere multiple (e.g., every, periodic, a majority) branch is apolypeptide (e.g., 2-2000 amino acids). In embodiments, the polypeptidepolymer is a polymer of a polymerized monomer attached to apolypeptide(e.g., 2-2000 amino acids) forming a brush polymer where multiple (e.g.,every, periodic, a majority) branches are a polypeptide (e.g., 2-2000amino acids). The term may include a polymerized polypeptide containingmaterial generated through direct polymerization of monomers including apolypeptide.

“Brush polymers,” as used herein are polymers of monomers (e.g.,monomers attached to a polypeptide that ultimately form a polymerizedmonomer). There are three main methodologies for the synthesis ofbrush-like polymeric architectures in general basis: the grafting onto,the grafting from, and the grafting through approach. The grafting ontoapproach is based on the attachment of already synthesized secondarychains onto a polymeric backbone with reactive sites usually randomlydistributed. In the grafting from approach, the polymer backbonecontaining initiating sites is used as macroinitiator for thepolymerization of a second monomer. In the grafting through approach, ormacromonomer approach, a polymer chain bearing a monomer unit ispolymerized with a second monomer to lead graft co-polymers. Inembodiments of the present disclosure, the brush polymers are preparedfrom the graft through polymerization of norbornyl-peptide monomers viaring opening metathesis polymerization (ROMP), using a catalystinitiator. In embodiments, the graft through polymerization ofnorbornyl-peptide monomers via ring opening metathesis polymerization(ROMP), using a catalyst initiator results in high density polypeptidepolymers with structures that resist proteolysis dependent on theirdegree of polymerization. In embodiments, the peptides in the highdensity brush polymers also maintain their intended biological function.In embodiments, the polymers described herein are not made using agrafting onto approach.

The term “graft-through polymerization” is used in accordance with itsplain meaning in the art of polymer chemistry and refers to amacromonomer method of graft polymer (e.g., polymer or copolymer)synthesis. The method forms the graft polymer (also referred to in thepolymer chemistry arts as the macromolecule) by polymerizingmacromonomer molecules having one polymerizable monomer end-group whichenables it to act as a monomer molecule, contributing a single monomericunit to a chain of the final macromolecule (graft polymer) (alsoreferred to herein as a polynmerized monomer). Unlike graft polymersassembled by graft-to (also commonly referred to as “graft-onto”)polymerization, graft polymers assembled by graft-through polymerizationdo not include unreacted functional groups (or chemical vestigesthereof) within the linear backbone used to graft side chain polymers tothe linear backbone. Graft-through polymerization includes methods ofpolymerization for synthesizing a polymer with monomer side chains(e.g., polypeptides) using a known polymerization strategy amenable tothe functional groups involved, including protected and unprotectedforms (e.g., of amino acid sidechains). Graft-through polymerization mayinclude synthesizing a polymer with monomer side chains (e.g.,polypeptide monomers) wherein the monomer side chains are not furtherderivatized or modified after synthesis of the polymer. Thus, graftpolymers assembled by graft-through polymerization include high densitypolymers having smaller distances between side chains relative to graftpolymers assembled by graft-to polymerization. In embodiments, the graftpolymer is a polypeptide polymer. In embodiments, the graft polymer is abrush polymer. In embodiments, the graft polymer synthesis is a brushpolymer synthesis. In embodiments, the graft-through polymerizationemploys atom transfer radical polymerization (ATRP), ring-openingmetathesis polymerization (ROMP), anionic and cationic polymerizations,free radical living polymerization, radiation-induced polymerization,ring-opening olefin metathesis polymerization, polycondensationreactions, or iniferter-induced polymerization.

“Cell penetrating,” when used to describe the various polypeptidepolymers or brush polymers disclosed herein, means polypeptide polymersor brush polymers that are capable of entering into the interior of acell. Canonical cell penetrating peptides (CPPs or CCPs, usedinterchangeably herein), are Tat (YGRKKRRQRRR, SEQ ID NO:2) and Arg8(RRRRRRRR, SEQ ID NO:3).

“Pendant,” when used to describe a peptide, an amino acid, or anon-peptidic moiety attached to a polypeptide polymers means, that thepeptide, the amino acid, or the non-peptidic moiety is covalently boundto a monomer (polymerized monomer) in the polymer. “High density” isbased on weight percentage of the pendant moiety in the polypeptidepolymer. For example a polypeptide polymer may be high density inpolypeptides (polypeptide moieties) when 100% of the constituentmonomers (polymerized monomers) are attached to a polypeptide(polypeptide moiety). In embodiments, a polypeptide polymer is highdensity when about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the constituent monomersare attached to a polypeptide. In embodiments, a polypeptide polymer ishigh density when greater than about 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of theconstituent monomers are attached to a polypeptide. In embodiments, apolypeptide polymer is high density when 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of theconstituent monomers are attached to a polypeptide. In embodiments, apolypeptide polymer is high density when greater than 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% of the constituent monomers are attached to a polypeptide. Inembodiments, a polypeptide polymer is high density when greater than 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99% of the constituent monomers are attached to apolypeptide. In embodiments, a polypeptide polymer is high density whengreater than 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of theconstituent monomers are attached to a polypeptide. In embodiments, apolypeptide polymer is high density when greater than 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% ofthe constituent monomers are attached to a polypeptide. In embodiments,a polypeptide polymer is high density when greater than 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% of the constituent monomers are attached to apolypeptide. In embodiments, a polypeptide polymer is high density whengreater than 95, 96, 97, 98, or 99% of the constituent monomers areattached to a polypeptide. In embodiments, a polypeptide polymer is highdensity when greater than 99% of the constituent monomers are attachedto a polypeptide.

As used herein, the term “living polymerization” is used in accordancewith its common meaning in the chemical sciences and refers to a form ofchain growth polymerization where the ability of a growing polymer chainto terminate has been removed.—Chain termination and chain transferreactions may be absent and the rate of chain initiation may be muchlarger than the rate of chain propagation. The result is that thepolymer chains may grow at a more constant rate than seen in traditionalchain polymerization and their lengths may remain very similar (i.e.they have a very low polydispersity index). Additional advantages mayinclude predetermined molar mass and control over end-groups.

The term “polymerizable monomer” is used in accordance with its meaningin the art of polymer chemistry and refers to a compound that maycovalently bind chemically to other monomer molecules to form a polymerthereby forming a polymerized monomer. An example of a polymerizablemonomer is a ROMP polymerizable monomer, which is a polymerizablemonomer capable of binding chemically to other ROMP polymerizablemonomers through a ROMP chemical reaction to form a polymer. It will beunderstood that a polymerizable monomer may be chemically modified inthe polymerization reaction to differ from the free polymerizablemonomer when forming the polymerized monomer moiety. In embodiments, theROMP polymerizable monomer includes an olefin. In embodiments, the ROMPpolymerizable monomer includes a cyclic olefin. In embodiments, the ROMPpolymerizable monomer includes a cyclic olefin with ring strain (e.g.,norbornene or cyclopentene or derivatives thereof). In embodiments, theROMP polymerizable monomer is attached to a polypeptide. In embodiments,the ROMP polymerizable monomer is attached to a hydrophobic moiety. Inembodiments, the ROMP polymerizable monomer is or includes a substitutedor unsubstituted norbornenyl. In embodiments, the ROMP polymerizablemonomer is

wherein Ring A is substituted or unsubstituted cycloalkyl, substitutedor unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R³ is independently apolypeptide, non-polypeptide, detectable moiety, therapeutic moiety,functional moiety, therapeutic polypeptide hydrophobic moiety, hydrogen,halogen, oxo, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OC(halo)₃,—OCH(halo)₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, R³ is independently a polypeptide. In embodiments, R³ isindependently a non-polypeptide. In embodiments, at least one R³ isindependently a polypeptide. In embodiments, R³ is independently ahalogen, oxo, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OC(halo)₃,—OCH(halo)₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, at least one R³ is independently a polypeptide. Inembodiments, R³ is independently a hydrogen, halogen, oxo, —C(halo)₃,—CH(halo)₂, —CH₂(halo), —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OC(halo)₃, —OCH(halo)₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

In embodiments, the ROMP polymerizable monomer is

In formula (IIA-IIF), Ring A, L¹, R³ and R⁴ are as defined herein. Inembodiments of formula (IIA)-(IIF), R³ is not a polypeptide. Inembodiments, the ROMP polymerizable monomer is

In formula (IIIA)-(IIIF), Ring A, L¹ and R⁴ is as defined herein.

L¹ is independently a bond, —O—, —NH—, —COO—, —S—, —SO₂—, —SO₃—, —SO₄—,—SO₂NH—, —NHC(O)—, —C(O)NH—, —NHC(O)O—, —NHC(O)NH—, C(O), substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In embodiments, L¹ isindependently a bond.

In embodiments, L¹ is independently a bond, —C(O)O—, —C(O)NH—,—C(O)NHCH₂CH₂NH—, —CH₂—, —CH₂CH₂O—, —CH₂CH₂—, —CH₂NHC(O)—,—CH₂CH₂NHC(O)—, —CH₂CH₂NH—, —CH₂O—, —CH₂CH₂N(CH₃)₂CH₂—, —CH₂C(O)—, or—CH₂CH₂C(O)—. In embodiments, L¹ is independently —C(O)O—, —C(O)NH—,—C(O)NHCH₂CH₂NH—, —CH₂—, —CH₂CH₂O—, —CH₂CH₂—, —CH₂NHC(O)—,—CH₂CH₂NHC(O)—, —CH₂CH₂NH—, —CH₂O—, —CH₂CH₂N(CH₃)₂CH₂—, —CH₂C(O)—, or—CH₂CH₂C(O)—. In embodiments, L¹ is independently —CH₂CH₂CH₂CH₂CH₂C(O)—.In embodiments, L¹ is independently —CH₂CH₂CH₂CH₂CH₂C(O)NH—. Inembodiments, L¹ is a cleavable linker. In embodiments, L¹ is cleavedupon entering a cell. In embodiments, L¹ is cleaved by the environmentinside a cell but not outside the cell. In embodiments, L¹ is cleaved bya change in pH. In embodiments, L¹ is cleaved by acidic pH. Inembodiments, L¹ is cleaved by the pH of a lysosome. In embodiments, L¹is cleaved by reducing conditions. In embodiments, L¹ is an enzymesubstrate and is cleaved by the enzyme. In embodiments, L¹ is cleavableby radiation. In embodiments, L¹ is light. In embodiments, L¹ includes aprodrug cleavable linkage.

R⁴ is independently a polypeptide, hydrogen, halogen, oxo, —C(halo)₃,—CH(halo)₂, —CH₂(halo), —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OC(halo)₃, —OCH(halo)₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁴ may independently be an amino acid orpolypeptide as described herein. R⁴ may be a polypeptide as describedherein. R⁴ may be an amino acid as described herein.

In embodiments, a polymerizable monomer is selected from:

The above polymerizable monomers form the polymerized monomers withinthe polymers disclosed herein.

A “terminal polymer moiety” as used herein, refers to a chemical moietythat results from termination of a polymerization reaction (e.g., byaddition of a chain terminator or transfer agent that may be modified toform the terminal polymer moiety in the termination reaction). Terminalpolymer moieties may include a solid support, nanoparticle orappropriate termination moiety (e.g. substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl or substituted or unsubstituted heteroaryl). Inembodiments, a terminal polymer moiety includes a functional moiety, forexample a detectable moiety. In embodiments, a terminal polymer moietyincludes a therapeutic moiety. In embodiments, a terminal polymer moietyincludes a label. In embodiments, a terminal polymer moiety is thepolymerization product of an ethyl vinyl ether. In embodiments, aterminal polymer moiety is the polymerization product of an alkenecontaining substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl. Inembodiments, a terminal polymer moiety is the polymerization product ofan alkene containing compound (e.g., also including a function group,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, therapeutic moiety, label, ordetectable moiety). In embodiments, a terminal polymer moiety isselected from:

The term “ring-opening metathesis polymerization” or “ROMP” is used inaccordance with its meaning in polymer chemistry and refers to achain-growth polymerization (e.g., olefin metathesis chain-growthpolymerization). In embodiments, the reaction is driven by relief ofring strain in cyclic olegins (e.g., norbornene or cyclopentene). Inembodiments, the ROMP uses a ruthenium catalyst. In embodiments, theROMP uses a Grubbs' catalyst. In embodiments, the ROMP uses a Mocatalyst. In embodiments, the ROMP uses [Mo(=CHBut)(Nar)(OR)2]. Inembodiments, the ROMP uses a transition metal catalyst. In embodiments,the ROMP uses a transition metal carbine complex catalyst. Inembodiments, the ROMP usesBenzylidene-bis(tricyclohexylphosphine)-dichlororuthenium. Inembodiments, the ROMP uses[1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(phenylmethylene)(tricyclohexylphosphine)ruthenium.In embodiments, the ROMP usesDichloro(o-isopropoxyphenylmethylene)(tricyclohexylphosphine)ruthenium(II).In embodiments, the ROMP uses[1,3-Bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro(o-isopropoxyphenylmethylene)ruthenium.In embodiments, the ROMP uses a third generation Grubbs' catalyst. Inembodiments, the ROMP uses (IMesH₂)(C₅H₅N)₂(Cl)₂Ru═CHPh.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchednon-cyclic carbon chain (or carbon), or combination thereof, which maybe fully saturated, mono- or polyunsaturated and can include di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkylgroup is one having one or more double bonds or triple bonds. Examplesof unsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. An alkoxy is an alkyl attached to theremainder of the molecule via an oxygen linker (—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms. Theterm “alkenylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable non-cyclic straight or branchedchain, or combinations thereof, including at least one carbon atom andat least one heteroatom selected from the group consisting of O, N, P,Si, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized, and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N, P, S, and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two or three heteroatoms may be consecutive, such as, forexample, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicnon-aromatic versions of “alkyl” and “heteroalkyl,” respectively,wherein the carbons making up the ring or rings do not necessarily needto be bonded to a hydrogen due to all carbon valencies participating inbonds with non-hydrogen atoms. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently (e.g., biphenyl). A fusedring aryl refers to multiple rings fused together wherein at least oneof the fused rings is an aryl ring. The term “heteroaryl” refers to arylgroups (or rings) that contain at least one heteroatom such as N, O, orS, wherein the nitrogen and sulfur atoms are optionally oxidized, andthe nitrogen atom(s) are optionally quaternized. Thus, the term“heteroaryl” includes fused ring heteroaryl groups (i.e., multiple ringsfused together wherein at least one of the fused rings is aheteroaromatic ring). A 5,6-fused ring heteroarylene refers to two ringsfused together, wherein one ring has 5 members and the other ring has 6members, and wherein at least one ring is a heteroaryl ring. Likewise, a6,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 6 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylenerefers to two rings fused together, wherein one ring has 6 members andthe other ring has 5 members, and wherein at least one ring is aheteroaryl ring. A heteroaryl group can be attached to the remainder ofthe molecule through a carbon or heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively.Non-limiting examples of heteroaryl groups include pyridinyl,pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl,benzodioxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyridinyl,indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl,benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl,benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl,imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl,pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl,isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl,tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl,pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl, or quinolyl. Theexamples above may be substituted or unsubstituted and divalent radicalsof each heteroaryl example above are non-limiting examples ofheteroarylene.

A fused ring heterocyloalkyl-aryl is an aryl fused to aheterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is aheteroaryl fused to a heterocycloalkyl. A fused ringheterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkylfused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl,fused ring heterocycloalkyl-heteroaryl, fused ringheterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substitutentsdescribed herein.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfonyl,” as used herein, means a moiety having theformula —S(O₂)—R′, where R′ is a substituted or unsubstituted alkylgroup as defined above. R′ may have a specified number of carbons (e.g.,“C₁-C₄ alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R″′, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)N R′R″,—NR″C(O)R′, —NR′—C(O)NR″R″′, —NR″C(O)₂R′, —NR—C(NR′R″R″′)═NR″ ″,—NR—C(NR′R″)═NR″′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R″′,—ONR′R″, —NR′C═(O)NR″NR″′R″ ″, —CN, —NO₂, in a number ranging from zeroto (2m′+1), where m′ is the total number of carbon atoms in suchradical. R, R′, R″, R″′, and R″ ″ each preferably independently refer tohydrogen, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl (e.g., aryl substituted with 1-3halogens), substituted or unsubstituted heteroaryl, substituted orunsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R″′, and R″ ″ group when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 4-, 5-, 6-, or 7-memberedring. For example, —NR′R″ includes, but is not limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R″′,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC (O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R″′, —NR″C(O)₂R′, —NR—C(NR′R″R″′)═NR″ ″, —NR—C(NR′R″)═NR″′,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R″′, —ONR′R″,—NR′C═(O)NR″NR″′R″ ″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on the aromatic ring system;and where R′, R″, R″′, and R″ ″ are preferably independently selectedfrom hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R″′,and R″ ″ groups when more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′— (C″R″R″′)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R″′ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,        —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl,        unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,            —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,            —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,            —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl,            unsubstituted heteroalkyl, unsubstituted cycloalkyl,            unsubstituted heterocycloalkyl, unsubstituted aryl,            unsubstituted heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,                —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,                —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H,                —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, heteroaryl, substituted with at least one                substituent selected from: oxo, halogen, —CF₃, —CN, —OH,                —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H,                —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂,                —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂,                —NHSO₂CH₃, —N₃, unsubstituted alkyl, unsubstituted                heteroalkyl, unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section below.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19(1977)). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts. Otherpharmaceutically acceptable carriers known to those of skill in the artare suitable for the present invention. Salts tend to be more soluble inaqueous or other protonic solvents that are the corresponding free baseforms. In other cases, the preparation may be a lyophilized powder in 1mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like)salts.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls. Moreover, where a moiety is substitutedwith an R substituent, the group may be referred to as “R-substituted.”Where a moiety is R-substituted, the moiety is substituted with at leastone R substituent and each R substituent is optionally different.

Descriptions of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The terms “treating” or “treatment” refers to any indicia of success inthe treatment or amelioration of an injury, disease, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; improving a patient's physical or mental well-being.The treatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation.

An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g. achieve the effect for which it is administered, treat adisease, reduce enzyme activity, increase enzyme activity, reducetranscriptional activity, increase transcriptional activity, reduce oneor more symptoms of a disease or condition). An example of an “effectiveamount” is an amount sufficient to contribute to the treatment,prevention, or reduction of a symptom or symptoms of a disease, whichcould also be referred to as a “therapeutically effective amount.” A“reduction” of a symptom or symptoms (and grammatical equivalents ofthis phrase) means decreasing of the severity or frequency of thesymptom(s), or elimination of the symptom(s). A “prophylacticallyeffective amount” of a drug is an amount of a drug that, whenadministered to a subject, will have the intended prophylactic effect,e.g., preventing or delaying the onset (or reoccurrence) of an injury,disease, pathology or condition, or reducing the likelihood of the onset(or reoccurrence) of an injury, disease, pathology, or condition, ortheir symptoms. The full prophylactic effect does not necessarily occurby administration of one dose, and may occur only after administrationof a series of doses. Thus, a prophylactically effective amount may beadministered in one or more administrations. An “activity decreasingamount,” as used herein, refers to an amount of antagonist (inhibitor)required to decrease the activity of an enzyme or protein (e.g.transcription factor) relative to the absence of the antagonist. An“activity increasing amount,” as used herein, refers to an amount ofagonist (activator) required to increase the activity of an enzyme orprotein (e.g. transcription factor) relative to the absence of theagonist. A “function disrupting amount,” as used herein, refers to theamount of antagonist (inhibitor) required to disrupt the function of anenzyme or protein (e.g. transcription factor) relative to the absence ofthe antagonist. A “function increasing amount,” as used herein, refersto the amount of agonist (activator) required to increase the functionof an enzyme or protein (e.g. transcription factor) relative to theabsence of the agonist. The exact amounts will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules, or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated, however, that the resulting reaction product can beproduced directly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture. The term “contacting” may includeallowing two species to react, interact, or physically touch, whereinthe two species may be a compound as described herein and a protein orenzyme. In some embodiments contacting includes allowing a compounddescribed herein to interact with a protein or enzyme that is involvedin a signaling pathway.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor (e.g. antagonist)interaction means negatively affecting (e.g. decreasing) the activity orfunction of the protein relative to the activity or function of theprotein in the absence of the inhibitor. In some embodiments inhibitionrefers to reduction of a disease or symptoms of disease. In someembodiments, inhibition refers to a reduction in the activity of asignal transduction pathway or signaling pathway. Thus, inhibitionincludes, at least in part, partially or totally blocking stimulation,decreasing, preventing, or delaying activation, or inactivating,desensitizing, or down-regulating signal transduction or enzymaticactivity or the amount of a protein.

As defined herein, the term “activation”, “activate”, “activating” andthe like in reference to a protein-activator (e.g. agonist) interactionmeans positively affecting (e.g. increasing) the activity or function ofthe protein

The term “modulator” refers to a composition that increases or decreasesthe level of a target molecule or the function of a target molecule.

“Patient” or “subject in need thereof” refers to a living organismsuffering from or prone to a disease or condition that can be treated byadministration of a compound or pharmaceutical composition, as providedherein. Non-limiting examples include humans, other mammals, bovines,rats, mice, dogs, monkeys, goat, sheep, cows, deer, and othernon-mammalian animals. In some embodiments, a patient is human. In someembodiments, a patient is a mammal. In some embodiments, a patient is amouse. In some embodiments, a patient is an experimental animal. In someembodiments, a patient is a rat. In some embodiments, a patient is atest animal.

“Disease” or “condition” refer to a state of being or health status of apatient or subject capable of being treated with a compound,pharmaceutical composition, or method provided herein.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intracranial, intranasal or subcutaneous administration, or theimplantation of a slow-release device, e.g., a mini-osmotic pump, to asubject. Administration is by any route, including parenteral andtransmucosal (e.g., buccal, sublingual, palatal, gingival, nasal,vaginal, rectal, or transdermal). In embodiments, administrationincludes direct administration to a tumor. Parenteral administrationincludes, e.g., intravenous, intramuscular, intra-arteriole,intradermal, subcutaneous, intraperitoneal, intraventricular, andintracranial. Other modes of delivery include, but are not limited to,the use of liposomal formulations, intravenous infusion, transdermalpatches, etc. By “co-administer” it is meant that a compositiondescribed herein is administered at the same time, just prior to, orjust after the administration of one or more additional therapies (e.g.anti-cancer agent or chemotherapeutic). The compound of the inventioncan be administered alone or can be coadministered to the patient.Coadministration is meant to include simultaneous or sequentialadministration of the compound individually or in combination (more thanone compound or agent). Thus, the preparations can also be combined,when desired, with other active substances (e.g. to reduce metabolicdegradation). The compositions of the present invention can be deliveredby transdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols. Oral preparations includetablets, pills, powder, dragees, capsules, liquids, lozenges, cachets,gels, syrups, slurries, suspensions, etc., suitable for ingestion by thepatient. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. Liquid formpreparations include solutions, suspensions, and emulsions, for example,water or water/propylene glycol solutions. The compositions of thepresent invention may additionally include components to providesustained release and/or comfort. Such components include high molecularweight, anionic mucomimetic polymers, gelling polysaccharides andfinely-divided drug carrier substrates. These components are discussedin greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and4,861,760. The entire contents of these patents are incorporated hereinby reference in their entirety for all purposes. The compositions of thepresent invention can also be delivered as microspheres for slow releasein the body. For example, microspheres can be administered viaintradermal injection of drug-containing microspheres, which slowlyrelease subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645,1995; as biodegradable and injectable gel formulations (see, e.g., GaoPharm. Res. 12:857-863, 1995); or, as microspheres for oraladministration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674,1997). In another embodiment, the formulations of the compositions ofthe present invention can be delivered by the use of liposomes whichfuse with the cellular membrane or are endocytosed, i.e., by employingreceptor ligands attached to the liposome, that bind to surface membraneprotein receptors of the cell resulting in endocytosis. By usingliposomes, particularly where the liposome surface carries receptorligands specific for target cells, or are otherwise preferentiallydirected to a specific organ, one can focus the delivery of thecompositions of the present invention into the target cells in vivo.(See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn,Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm.46:1576-1587, 1989). The compositions of the present invention can alsobe delivered as nanoparticles.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents used herein means at least two nucleotides covalently linkedtogether. The term “nucleic acid” includes single-, double-, ormultiple-stranded DNA, RNA and analogs (derivatives) thereof.Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25,30, 40, 50 or more nucleotides in length, up to about 100 nucleotides inlength. Nucleic acids and polynucleotides are a polymers of any length,including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000,7000, 10,000, etc. In certain embodiments. the nucleic acids hereincontain phosphodiester bonds. In other embodiments, nucleic acid analogsare included that may have alternate backbones (e.g. phosphodiesterderivatives), including, e.g., phosphoramidate, phosphorodiamidate,phosphorothioate (also known as phosphothioate), phosphorodithioate,phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid,phosphonoformic acid, methyl phosphonate, boron phosphonate, peptidenucleic acid linkages, or O-methylphosphoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages.Other analog nucleic acids include those with positive backbones;non-ionic backbones, modified sugars, and non-ribose backbones (e.g.phosphorodiamidate morpholino oligos or locked nucleic acids (LNA)),including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, andChapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modificationsin Antisense Research, Sanghui & Cook, eds. Nucleic acids containing oneor more carbocyclic sugars are also included within one definition ofnucleic acids. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments or as probeson a biochip. Mixtures of naturally occurring nucleic acids and analogscan be made; alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. In embodiments, the internucleotide linkages in DNA arephosphodiester, phosphodiester

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical,magnetic resonance imaging, or other physical means. For example, usefuldetectable moieties include peptide tags, protein tags, ³²P, fluorescentdyes, electron-dense reagents, enzymes (e.g., as commonly used in anELISA), biotin, digoxigenin, paramagnetic molecules, paramagneticnanoparticles, ultrasmall superparamagnetic iron oxide (“USPIO”)nanoparticles, USPIO nanoparticle aggregates, superparamagnetic ironoxide (“SPIO”) nanoparticles, SPIO nanoparticle aggregates,monochrystalline SPIO, monochrystalline SPIO aggregates,monochrystalline iron oxide nanoparticles, monochrystalline iron oxide,other nanoparticle contrast agents, liposomes or other delivery vehiclescontaining Gadolinium chelate (“Gd-chelate”) molecules, Gadolinium,radioisotopes, radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15,fluorine-18, rubidium-82), fluorodeoxyglucose (e.g. fluorine-18labeled), any gamma ray emitting radionuclides, positron-emittingradionuclide, radiolabeled glucose, radiolabeled water, radiolabeledammonia, biocolloids, microbubbles (e.g. including microbubble shellsincluding albumin, galactose, lipid, and/or polymers; microbubble gascore including air, heavy gas(es), perfluorcarbon, nitrogen,octafluoropropane, perflexane lipid microsphere, perflutren, etc.),iodinated contrast agents (e.g. iohexol, iodixanol, ioversol, iopamidol,ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate,thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates,fluorophores, two-photon fluorophores, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget peptide. Detectable moieties also include any of the abovecompositions encapsulated in nanoparticles, particles, aggregates,coated with additional compositions, derivatized for binding to atargeting agent (e.g. compound described herein). Any method known inthe art for conjugating an oligonucleotide or protein to the label maybe employed, e.g., using methods described in Hermanson, BioconjugateTechniques 1996, Academic Press, Inc., San Diego.

MRI can be used to non-invasively acquire tissue images with highresolution. Paramagnetic agents or USPIO nanoparticles or aggregatesthereof enhance signal attenuation on T₂-weighted magnetic resonanceimages, and conjugation of such nanoparticles to binding ligands permitsthe detection of specific molecules at the cellular level. For example,MRI with nanoparticle detection agents can detect small foci of cancer.See e.g., Y. W. Jun et al., 2005, J. Am. Chern. Soc. 127:5732-5733; Y.M. Huh et al., 2005, J. Am. Chern. Soc. 127:12387-12391.Contrast-enhanced MRI is well-suited for the dynamic non-invasiveimaging of macromolecules or of molecular events, but it requiresligands that specifically bind to the molecule of interest. J. W. Bulteet al., 2004, NMR Biomed. 17:484-499. Fluorescent dyes and fluorophores(e.g. fluorescein, fluorescein isothiocyanate, and fluoresceinderivatives) can be used to non-invasively acquire tissue images withhigh resolution, with for example spectrophotometry, two-photonfluorescence, two-photon laser microscopy, or fluorescence microscopy(e.g. of tissue biopsies). MRI can be used to non-invasively acquiretissue images with high resolution, with for example paramagneticmolecules, paramagnetic nanoparticles, ultrasmall superparamagnetic ironoxide (“USPIO”) nanoparticles, USPIO nanoparticle aggregates,superparamagnetic iron oxide (“SPIO”) nanoparticles, SPIO nanoparticleaggregates, monochrystalline iron oxide nanoparticles, monochrystallineiron oxide, other nanoparticle contrast agents. MRI can be used tonon-invasively acquire tissue images with high resolution, with forexample Gadolinium, including liposomes or other delivery vehiclescontaining Gadolinium chelate (“Gd-chelate”) molecules. Positronemission tomography (PET), PET/computed tomography (CT), single photonemission computed tomography (SPECT), and SPECT/CT can be used tonon-invasively acquire tissue images with high resolution, with forexample radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15,fluorine-18, rubidium-82), fluorodeoxyglucose (e.g. fluorine-18labeled), any gamma ray emitting radionuclides, positron-emittingradionuclide, radiolabeled glucose, radiolabeled water, radiolabeledammonia. Ultrasound (ultrasonography) and contrast enhanced ultrasound(contrast enhanced ultrasonography) can be used to non-invasivelyacquire tissue images with high resolution, with for example biocolloidsor microbubbles (e.g. including microbubble shells including albumin,galactose, lipid, and/or polymers; microbubble gas core including air,heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexanelipid microsphere, perflutren, etc.). X-ray imaging (radiography) or CTcan be used to non-invasively acquire tissue images with highresolution, with for example iodinated contrast agents (e.g. iohexol,iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate,metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, goldnanoparticles, or gold nanoparticle aggregates. These detection methodsand instruments and detectable moieties capable of being measured ordetected by the corresponding method are non-limiting examples.

In embodiments, the peptide tags are: AviTag, a peptide allowingbiotinylation by the enzyme BirA and so the protein can be isolated bystreptavidin (GLNDIFEAQKIEWHE, SEQ ID NO:25), Calmodulin-tag, a peptidebound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL, SEQ IDNO:26), polyglutamate tag, a peptide binding efficiently toanion-exchange resin such as Mono-Q (EEEEEE, SEQ ID NO:27), E-tag, apeptide recognized by an antibody (GAPVPYPDPLEPR, SEQ ID NO:28),FLAG-tag, a peptide recognized by an antibody (DYKDDDDK, SEQ ID NO:29),HA-tag, a peptide recognized by an antibody (YPYDVPDYA, SEQ ID NO:30),His-tag, 5-10 histidines bound by a nickel or cobalt chelate (HHHHHH,SEQ ID NO:31), Myc-tag, a short peptide recognized by an antibody(EQKLISEEDL, SEQ ID NO:32), S-tag (KETAAAKFERQHMDS, SEQ ID NO:33),SBP-tag, a peptide which binds to streptavidin(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP, SEQ ID NO:34), Softag 1, formammalian expression (SLAELLNAGLGGS, SEQ ID NO:35), Softag 3, forprokaryotic expression (TQDPSRVG, SEQ ID NO:36), Strep-tag, a peptidewhich binds to streptavidin or the modified streptavidin calledstreptactin (Strep-tag II: WSHPQFEK, SEQ ID NO:37), TC tag, atetracysteine tag that is recognized by FlAsH and ReAsH biarsenicalcompounds (CCPGCC. SEQ ID NO:38), V5 tag, a peptide recognized by anantibody (GKPIPNPLLGLDST, SEQ ID NO:39), VSV-tag, a peptide recognizedby an antibody (YTDIEMNRLGK, SEQ ID NO:40), Xpress tag (DLYDDDDK, SEQ IDNO:41). In embodiments, covalent peptide tags are: Isopeptag, a peptidewhich binds covalently to pilin-C protein (TDKDMTITFTNKKDAE, SEQ IDNO:42), SpyTag, a peptide which binds covalently to SpyCatcher protein(AHIVMVDAYKPTK, SEQ ID NO:43). In embodiments, protein tags are: BCCP(Biotin Carboxyl Carrier Protein), a protein domain biotinylated by BirAenabling recognition by streptavidin, Glutathione-S-transferase-tag, aprotein which binds to immobilized glutathione, Green fluorescentprotein-tag, a protein which is spontaneously fluorescent and can bebound by nanobodies, Halo-tag, a mutated hydrolase that covalentlyattaches to the HaloLink™ Resin (Promega), Maltose binding protein-tag,a protein which binds to amylose agarose, Nus-tag, Thioredoxin-tag,Fc-tag, derived from immunoglobulin Fc domain, allow dimerization andsolubilization.

“Therapeutic moiety” as used herein refers to a monovalent agent capableof treating a disease. In embodiments, the therapeutic moiety may beactive when included in a graft polymer (e.g, polypeptide polymer). Inembodiments, the therapeutic moiety may be active when released from thegraft polymer (e.g., polypeptide polymer) as a therapeutic agent, forexample through a chemical reaction. In embodiments, the activetherapeutic agent is modified from the therapeutic moiety through theaddition of a chemical moiety or removal of a portion of the therapeuticmoiety during the release reaction. Examples of therapeutic agents ormoieties include anti-cancer agents, cytotoxic agents, cytostaticagents, anti-inflammatory agents, analgesics, anti-infective agents,growth inhibitory agent; cytotoxic agents; immunogenic agent;immunomodulatory agents; agents that modulate T-cell activity;chemokines. A “therapeutic polypeptide” is a polypeptide capable oftreating a disease.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

As used herein, the term “conjugated” when referring to two moietiesmeans the two moieties are bonded, wherein the bond or bonds connectingthe two moieties may be covalent or non-covalent. In embodiments, thetwo moieties are covalently bonded to each other (e.g. directly orthrough a covalently bonded intermediary). In embodiments, the twomoieties are non-covalently bonded (e.g. through ionic bond(s), van derwaal's bond(s)/interactions, hydrogen bond(s), polar bond(s), orcombinations or mixtures thereof).

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments,about means within a standard deviation using measurements generallyacceptable in the art. In embodiments, about means a range extending to+/−10% of the specified value. In embodiments, about means the specifiedvalue.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature. Examples of amino acid mimetics and polypeptidemimetics include peptoids, D-peptides, and 3-peptides. Amino acids maybe modified amino acids (natural or mimetics) including additionalmoieties, for example function, therapeutic, or detectable moieties.Modified amino acids may be modified in the side chain by the additionof additional moieties.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

Twenty amino acids are commonly found in proteins. Those amino acids canbe grouped into nine classes or groups based on the chemical propertiesof their side chains. Substitution of one amino acid residue for anotherwithin the same class or group is referred to herein as a “conservative”substitution. Conservative amino acid substitutions can frequently bemade in a protein without significantly altering the conformation orfunction of the protein. Substitution of one amino acid residue foranother from a different class or group is referred to herein as a“non-conservative” substitution. In contrast, non-conservative aminoacid substitutions tend to modify conformation and function of aprotein.

Example of Amino Acid Classification

Small/Aliphatic residues: Gly, Ala, Val, Leu, Ile Cyclic Imino Acid: ProHydroxyl-containing Residues: Ser, Thr Acidic Residues: Asp, Glu AmideResidues: Asn, Gln Basic Residues: Lys, Arg Imidazole Residue: HisAromatic Residues: Phe, Tyr, Trp Sulfur-containing Residues: Met, Cys

In some embodiments, the conservative amino acid substitution comprisessubstituting any of glycine (G), alanine (A), isoleucine (I), valine(V), and leucine (L) for any other of these aliphatic amino acids;serine (S) for threonine (T) and vice versa; aspartic acid (D) forglutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) andvice versa; lysine (K) for arginine (R) and vice versa; phenylalanine(F), tyrosine (Y) and tryptophan (W) for any other of these aromaticamino acids; and methionine (M) for cysteine (C) and vice versa. Othersubstitutions can also be considered conservative, depending on theenvironment of the particular amino acid and its role in thethree-dimensional structure of the protein. For example, glycine (G) andalanine (A) can frequently be interchangeable, as can alanine (A) andvaline (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pKs of these two amino acid residues arenot significant. Still other changes can be considered “conservative” inparticular environments (see, e.g., BIOCHEMISTRY at pp. 13-15, 2nd ed.Lubert Stryer ed. (Stanford University); Henikoff et al., Proc. Nat'lAcad. Sci. USA (1992) 89:10915-10919; Lei et al., J. Biol. Chem. (1995)270(20): 11882-11886).

“Polypeptide,” “peptide,” and “protein” are used herein interchangeablyand mean any peptide-linked chain of amino acids, regardless of lengthor post-translational modification. As noted below, the polypeptidesdescribed herein can be, e.g., wild-type proteins, biologically-activefragments of the wild-type proteins, or variants of the wild-typeproteins or fragments. Variants, in accordance with the disclosure, cancontain amino acid substitutions, deletions, or insertions. Thesubstitutions can be conservative or non-conservative. The terms applyto amino acid polymers in which one or more amino acid residue is anartificial chemical mimetic of a corresponding naturally occurring aminoacid, as well as to naturally occurring amino acid polymers andnon-naturally occurring amino acid polymer. When a polypeptide includesamino acid mimetics or modified amino acids, the monomer may beconnected through bonds that are different from or derivatives ofpeptide links.

“Isolated,” when used to describe the various polypeptides disclosedherein, means a polypeptide that has been identified and separatedand/or recovered from a component of its natural environment forexample, that has been separated or purified from components (e.g.,proteins or other naturally-occurring biological or organic molecules)which naturally accompany it, e.g., other proteins, lipids, and nucleicacid in a cell expressing the proteins. Typically, a polypeptide ispurified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80,85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in asample. Contaminant components of its natural environment are materialsthat would typically interfere with diagnostic or therapeutic uses forthe polypeptide, and may include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In embodiments, thepolypeptide may be purified (1) to a degree sufficient to obtain atleast 15 residues of N-terminal or internal amino acid sequence by useof a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the polypeptidenatural environment will not be present. In embodiments, isolatedpolypeptide may be prepared by at least one purification step.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions.

“Active” or “activity” for the purposes herein refers to form(s) of apolypeptide, which retain a biological and/or an immunological activityof native or naturally-occurring form of that polypeptide, where“biological” activity refers to a biological function (either inhibitoryor stimulatory) caused by a native or naturally-occurring polypeptide.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all. Transgenic cells and plants are thosethat express a heterologous gene or coding sequence, typically as aresult of recombinant methods.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects. In some embodiments, acontrol is the measurement of the activity of a protein in the absenceof a compound as described herein (including embodiments and examples).

Composition

In an aspect is provided a polymer having the formula: R¹-[M(O)]_(n)—R²wherein, n is an integer from 2 to 1000; M is a polymerized monomer; Ois independently a polypeptide (a polypeptide moiety) covalentlyattached (e.g., bonded) to M through a covalent linker; and R¹ and R²are independently terminal polymer moieties.

In an aspect is provided a block copolymer having the formula:R¹-[M(O)]_(n)-[M(P)]_(m)—R² or R¹-[M(P)]_(m)-[M(O)]_(n)—R² wherein, n isan integer from 2 to 1000; m is an integer from 2 to 1000; M is apolymerized monomer; O is independently a polypeptide (a polypeptidemoiety) covalently attached (e.g., bonded) to M through a covalentlinker (a polypeptide moiety); P is independently a non-polypeptidemoiety; and R¹ and R² are independently terminal polymer moieties.

In an aspect is provided a blend copolymer having the formula:R¹-([M(O)]_(n)-[M(P)]_(m))_(z)—R² or R¹-([M(P)]_(m)-[M(O)]_(n))_(z)—R²wherein, n is an integer from 2 to 1000; m is an integer from 2 to 1000;M is a polymerized monomer; O is independently a polypeptide (apolypeptide moiety) covalently attached (e.g., bonded) to M through acovalent linker; P is independently a non-polypeptide moiety; z is aninteger from 2 to 100; and R¹ and R² are independently terminal polymermoieties.

In embodiments, O is covalently bonded to M (polymerizable monomer orpolymerized monomer) through a covalent linker. In embodiments, O iscovalently bonded to M through a covalent bond. O may also be referredto herein as R³ as described herein. In embodiments, O is an -L¹-R⁴moiety as described herein. In embodiments, O is an -L¹-R⁴ moietywherein L¹ is a covalent bond.

In embodiments, the polymer or copolymer includes a linear backbonecomprising a polymer of M units (the polymerized monomers). Inembodiments, the linear backbone is a polynorbomyl chain (composed ofnorbomyl polymerized monomers as described herein). In embodiments, thelinear backbone is a polynorbornyl derivative chain. In embodiments, thelinear backbone is a poly-substituted norbornyl chain. In embodiments,the linear backbone is a substituted polynorbornene. In embodiments, thelinear backbone is a polynorbornene. In embodiments, the linear backboneis a polynorbornene substituted with oligonucleotides at each norbornenemonomer. In embodiments, the linear backbone is a polynorbornenesubstituted with a polypeptide, non-polypeptide, detectable moiety,therapeutic moiety, functional moiety, therapeutic polypeptidehydrophobic moiety, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl substituted or unsubstituted heteroaryl, or acombination thereof. In embodiments, the linear backbone (e.g, polyM) ispolymerized from monomers of:

wherein Ring A, n and R³ (O or P) are as set forth herein. Inembodiments, the linker backbone is polymerized from monomers of:

In formula (IIA)-(IIF), Ring A, R³ (O or P), R⁴, and L¹ are as set forthherein. In embodiments, the linker backbone is polymerized from monomersof:

In formula (IIIA)-(IIIF), Ring A, L¹ and R4 is as defined herein. Inembodiments, M(O) is

In embodiments, M(O) is

In embodiments, M(O) is

In embodiments, M(O) is

In embodiments, M(O) is

In embodiments, M(O) is

In the M(O) monomers immediately above, the polypeptide is bonded to theC(O) through the peptide backbone nitrogen when the polypeptide includenaturally occurring amino acids (e.g., 20 natural amino acids). Inembodiments, each polypeptide (O, and alternatively referred to hereinas R³) in the polymer is optionally different. In embodiments, eachpolypeptide (e.g. O; R³ or R⁴) in the polymer is identical. Inembodiments, the polymer includes blocks of polypeptide (e.g. O; R³ orR⁴) wherein the polypeptides in each block are identical and thepolypeptide (e.g. O; R³ or R⁴) in different blocks are optionallydifferent. In embodiments, the polymer includes blocks of polypeptide(e.g. O; R³ or R⁴) wherein the polypeptides in each block are identicaland the polypeptide (e.g. O; R³ or R⁴) in different blocks aredifferent.

In embodiments, Ring A is independently substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R³ isindependently a polypeptide, non-polypeptide, detectable moiety,therapeutic moiety, functional moiety, therapeutic polypeptidehydrophobic moiety, hydrogen, halogen, oxo, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OC(halo)₃, —OCH(halo)₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. In embodiments, R³ is independently a polypeptide. Inembodiments, at least one R³ is independently a polypeptide. Inembodiments, R³ is independently a halogen, oxo, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OC(halo)₃, —OCH(halo)₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. In embodiments, at least one R³ is independently apolypeptide. In embodiments, R³ is independently a hydrogen, halogen,oxo, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OC(halo)₃,—OCH(halo)₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

In embodiments, L¹ is independently a bond, —O—, —NH—, —COO—, —S—,—SO₂—, —SO₃—, —SO₄—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —NHC(O)O—, —NHC(O)NH—,—C(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene. In embodiments, L¹ is independently a bond, —C(O)O—,—C(O)NH—, —C(O)NHCH₂CH₂NH—, —CH₂—, —CH₂CH₂O—, —CH₂CH₂—, —CH₂NHC(O)—,—CH₂CH₂NHC(O)—, —CH₂CH₂NH—, —CH₂O—, —CH₂CH₂N(CH₃)₂CH₂—, —CH₂C(O)—, or—CH₂CH₂C(O)—. In embodiments, L¹ is independently —C(O)O—, —C(O)NH—,—C(O)NHCH₂CH₂NH—, —CH₂—, —CH₂CH₂O—, —CH₂CH₂—, —CH₂NHC(O)—,—CH₂CH₂NHC(O)—, —CH₂CH₂NH—, —CH₂O—, —CH₂CH₂N(CH₃)₂CH₂—, —CH₂C(O)—, or—CH₂CH₂C(O)—. In embodiments, L¹ is independently —CH₂CH₂CH₂CH₂CH₂C(O)—.In embodiments, L¹ is independently —CH₂CH₂CH₂CH₂CH₂C(O)NH—. Inembodiments, L¹ is a cleavable linker. In embodiments, L¹ is cleavedupon entering a cell. In embodiments, L¹ is cleaved by the environmentinside a cell but not outside the cell. In embodiments, L¹ is cleaved bya change in pH. In embodiments, L¹ is cleaved by acidic pH. Inembodiments, L¹ is cleaved by the pH of a lysosome. In embodiments, L¹is cleaved by reducing conditions. In embodiments, L¹ is an enzymesubstrate and is cleaved by the enzyme. In embodiments, L¹ is cleavableby radiation. In embodiments, L¹ is light. In embodiments, L¹ includes aprodrug cleavable linkage.

In embodiments, R⁴ is independently a polypeptide, non-polypeptidemoiety, detectable moiety, therapeutic moiety, functional moiety,therapeutic polypeptide hydrophobic moiety, hydrogen, halogen, oxo,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂,—NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OC(halo)₃, —OCH(halo)₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R⁴ may independently be anamino acid or polypeptide as described herein. R⁴ may be a polypeptideas described herein. R⁴ may be an amino acid as described herein.

In embodiments, the polymerized monomer isN-substituted-5-norbornene-2,3-dicarboximide, wherein the substitutioncomprises the therapeutic polypeptide. In embodiments, M(O) is

L¹R⁴; L¹ is is independently a bond, —O—, —NH—, —COO—, —S—, —SO₂—,—SO₃—, —SO₄—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —NHC(O)O—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and R⁴ is a therapeuticpolypeptide. In embodiments, M(O) is

L¹ is is independently a bond, —O—, —NH—, —COO—, —S—, —SO₂—, —SO₃—,—SO₄—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —NHC(O)O—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and R⁴ is a polypeptide.

In embodiments, n is an integer from 2 to 2000. In embodiments, n is aninteger from 2 to 1000. In embodiments, n is an integer from 2 to 900.In embodiments, n is an integer from 2 to 800. In embodiments, n is aninteger from 2 to 700. In embodiments, n is an integer from 2 to 600. Inembodiments, n is an integer from 2 to 500. In embodiments, n is aninteger from 2 to 400. In embodiments, n is an integer from 2 to 300. Inembodiments, n is an integer from 2 to 200. In embodiments, n is aninteger from 2 to 100. In embodiments, n is an integer from 2 to 50. Inembodiments, n is an integer from 2 to 49. In embodiments, n is aninteger from 2 to 48. In embodiments, n is an integer from 2 to 47. Inembodiments, n is an integer from 2 to 46. In embodiments, n is aninteger from 2 to 45. In embodiments, n is an integer from 2 to 44. Inembodiments, n is an integer from 2 to 43. In embodiments, n is aninteger from 2 to 42. In embodiments, n is an integer from 2 to 41. Inembodiments, n is an integer from 2 to 40. In embodiments, n is aninteger from 2 to 39. In embodiments, n is an integer from 2 to 38. Inembodiments, n is an integer from 2 to 37. In embodiments, n is aninteger from 2 to 36. In embodiments, n is an integer from 2 to 35. Inembodiments, n is an integer from 2 to 34. In embodiments, n is aninteger from 2 to 33. In embodiments, n is an integer from 2 to 32. Inembodiments, n is an integer from 2 to 31. In embodiments, n is aninteger from 2 to 30. In embodiments, n is an integer from 2 to 29. Inembodiments, n is an integer from 2 to 28. In embodiments, n is aninteger from 2 to 27. In embodiments, n is an integer from 2 to 26. Inembodiments, n is an integer from 2 to 25. In embodiments, n is aninteger from 2 to 24. In embodiments, n is an integer from 2 to 23. Inembodiments, n is an integer from 2 to 22. In embodiments, n is aninteger from 2 to 21. In embodiments, n is an integer from 2 to 20. Inembodiments, n is an integer from 2 to 19. In embodiments, n is aninteger from 2 to 18. In embodiments, n is an integer from 2 to 17. Inembodiments, n is an integer from 2 to 16. In embodiments, n is aninteger from 2 to 15. In embodiments, n is an integer from 2 to 14. Inembodiments, n is an integer from 2 to 13. In embodiments, n is aninteger from 2 to 12. In embodiments, n is an integer from 2 to 11. Inembodiments, n is an integer from 2 to 10. In embodiments, n is aninteger from 2 to 9. In embodiments, n is an integer from 2 to 8. Inembodiments, n is an integer from 2 to 7. In embodiments, n is aninteger from 2 to 6. In embodiments, n is an integer from 2 to 5. Inembodiments, n is an integer from 2 to 4. In embodiments, n is aninteger from 2 to 3. In embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506,507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534,535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548,549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562,563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576,577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590,591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604,605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618,619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632,633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660,661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674,675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688,689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716,717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730,731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758,759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772,773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786,787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800,801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814,815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828,829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842,843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856,857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870,871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884,885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898,899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912,913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926,927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940,941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954,955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968,969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982,983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996,997, 998, 999, or 1000.

In an embodiment the polypeptide is a therapeutic polypeptide.Non-limiting examples of a therapeutic polypeptide include: a cellgrowth or proliferation inhibitory peptide, an anti-inflammatorypeptide, an anti-tumor or anti-cancer peptide, an anti-apoptoticpeptide, anti-diabetic, anti-obesity, anti-infective, anti-bacterial,anti-viral, peptides for promoting cell growth and differentiation,peptides for preventing pain, and peptides for preventing or treatingneural degeneration and/or peptides for promoting neurogenesis.

In embodiments, the polymer includes at least 5 polypeptide (e.g. O; R³or R⁴) branches. In embodiments, the polymer includes at least 10polypeptide (e.g. O; R³ or R⁴) branches. In embodiments, the polymerincludes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510,511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538,539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552,553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580,581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608,609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636,637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664,665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678,679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692,693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720,721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734,735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748,749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762,763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776,777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790,791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804,805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832,833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846,847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860,861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874,875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888,889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902,903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916,917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930,931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944,945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958,959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972,973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986,987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000polypeptide (e.g. O, R³ or R⁴) branches.

In embodiments, the polypeptide (e.g. O; R³ or R⁴) is from 5 to 2000amino acids long. In embodiments, the polypeptide (e.g. O; R³ or R⁴) isfrom 5 to 1000 amino acids long. In embodiments, the polypeptide is from5 to 900 amino acids long. In embodiments, the polypeptide is from 5 to800 amino acids long. In embodiments, the polypeptide is from 5 to 700amino acids long. In embodiments, the polypeptide is from 5 to 600 aminoacids long. In embodiments, the polypeptide is from 5 to 500 amino acidslong. In embodiments, the polypeptide is from 5 to 400 amino acids long.In embodiments, the polypeptide is from 5 to 300 amino acids long. Inembodiments, the polypeptide is from 5 to 200 amino acids long. Inembodiments, the polypeptide is from 5 to 100 amino acids long. Inembodiments, the polypeptide is from 5 to 50 amino acids long. Inembodiments, the polypeptide is from 5 to 49 amino acids long. Inembodiments, the polypeptide is from 5 to 48 amino acids long. Inembodiments, the polypeptide is from 5 to 47 amino acids long. Inembodiments, the polypeptide is from 5 to 46 amino acids long. Inembodiments, the polypeptide is from 5 to 45 amino acids long. Inembodiments, the polypeptide is from 5 to 44 amino acids long. Inembodiments, the polypeptide is from 5 to 43 amino acids long. Inembodiments, the polypeptide is from 5 to 42 amino acids long. Inembodiments, the polypeptide is from 5 to 41 amino acids long. Inembodiments, the polypeptide is from 5 to 40 amino acids long. Inembodiments, the polypeptide is from 5 to 39 amino acids long. Inembodiments, the polypeptide is from 5 to 38 amino acids long. Inembodiments, the polypeptide is from 5 to 37 amino acids long. Inembodiments, the polypeptide is from 5 to 36 amino acids long. Inembodiments, the polypeptide is from 5 to 35 amino acids long. Inembodiments, the polypeptide is from 5 to 34 amino acids long. Inembodiments, the polypeptide is from 5 to 33 amino acids long. Inembodiments, the polypeptide is from 5 to 32 amino acids long. Inembodiments, the polypeptide is from 5 to 31 amino acids long. Inembodiments, the polypeptide is from 5 to 30 amino acids long. Inembodiments, the polypeptide is from 5 to 29 amino acids long. Inembodiments, the polypeptide is from 5 to 28 amino acids long. Inembodiments, the polypeptide is from 5 to 27 amino acids long. Inembodiments, the polypeptide is from 5 to 26 amino acids long. Inembodiments, the polypeptide is from 5 to 25 amino acids long. Inembodiments, the polypeptide is from 5 to 24 amino acids long. Inembodiments, the polypeptide is from 5 to 23 amino acids long. Inembodiments, the polypeptide is from 5 to 22 amino acids long. Inembodiments, the polypeptide is from 5 to 21 amino acids long. Inembodiments, the polypeptide is from 5 to 20 amino acids long. Inembodiments, the polypeptide is from 5 to 19 amino acids long. Inembodiments, the polypeptide is from 5 to 18 amino acids long. Inembodiments, the polypeptide is from 5 to 17 amino acids long. Inembodiments, the polypeptide is from 5 to 16 amino acids long. Inembodiments, the polypeptide is from 5 to 15 amino acids long. Inembodiments, the polypeptide is from 5 to 14 amino acids long. Inembodiments, the polypeptide is from 5 to 13 amino acids long. Inembodiments, the polypeptide is from 5 to 12 amino acids long. Inembodiments, the polypeptide is from 5 to 11 amino acids long. Inembodiments, the polypeptide is from 5 to 10 amino acids long. Inembodiments, the polypeptide is from 5 to 9 amino acids long. Inembodiments, the polypeptide is from 5 to 8 amino acids long. Inembodiments, the polypeptide is from 5 to 7 amino acids long. Inembodiments, the polypeptide is from 5 to 6 amino acids long. Inembodiments, the polypeptide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563,564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577,578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591,592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605,606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619,620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633,634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647,648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661,662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675,676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689,690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703,704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717,718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731,732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745,746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759,760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773,774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787,788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801,802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815,816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829,830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843,844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857,858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871,872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885,886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899,900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913,914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927,928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941,942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955,956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969,970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983,984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997,998, 999, or 1000 amino acids long.

In embodiments, the polypeptide includes one (e.g. no more than one)arginine amino acid. In embodiments, the polypeptide includes two (e.g.no more than two) arginine amino acids. In embodiments, the polypeptideincludes three (e.g. no more than three) arginine amino acids. Inembodiments, the polypeptide includes four (e.g. no more than four)arginine amino acids. In embodiments, the polypeptide includes five(e.g. no more than five) arginine amino acids. In embodiments, thepolypeptide includes six (e.g. no more than six) arginine amino acids.In embodiments, the polypeptide includes from 1 to 6 arginine aminoacids. In embodiments, the polypeptide includes from 1 to 5 arginineamino acids. In embodiments, the polypeptide includes from 1 to 4arginine amino acids. In embodiments, the polypeptide includes from 1 to3 arginine amino acids. In embodiments, the polypeptide includes from 1to 2 arginine amino acids. In embodiments, the polypeptide includes from2 to 5 arginine amino acids. In embodiments, the polypeptide includesfrom 2 to 4 arginine amino acids. In embodiments, the polypeptideincludes from 2 to 3 arginine amino acids. In embodiments, thepolypeptide includes from 3 to 6 arginine amino acids. In embodiments,the polypeptide includes from 3 to 5 arginine amino acids. Inembodiments, the polypeptide includes from 3 to 4 arginine amino acids.In embodiments, the polypeptide includes from 4 to 6 arginine aminoacids. In embodiments, the polypeptide includes from 4 to 5 arginineamino acids. In embodiments, the polypeptide includes from 5 to 6arginine amino acids.

In embodiments, the polypeptide includes one (e.g. no more than one)lysine amino acid. In embodiments, the polypeptide includes two (e.g. nomore than two) lysine amino acids. In embodiments, the polypeptideincludes three (e.g. no more than three) lysine amino acids. Inembodiments, the polypeptide includes four (e.g. no more than four)lysine amino acids. In embodiments, the polypeptide includes five (e.g.no more than five) lysine amino acids. In embodiments, the polypeptideincludes six (e.g. no more than six) lysine amino acids. In embodiments,the polypeptide includes from 1 to 6 lysine amino acids. In embodiments,the polypeptide includes from 1 to 5 lysine amino acids. In embodiments,the polypeptide includes from 1 to 4 lysine amino acids. In embodiments,the polypeptide includes from 1 to 3 lysine amino acids. In embodiments,the polypeptide includes from 1 to 2 lysine amino acids. In embodiments,the polypeptide includes from 2 to 5 lysine amino acids. In embodiments,the polypeptide includes from 2 to 4 lysine amino acids. In embodiments,the polypeptide includes from 2 to 3 lysine amino acids. In embodiments,the polypeptide includes from 3 to 6 lysine amino acids. In embodiments,the polypeptide includes from 3 to 5 lysine amino acids. In embodiments,the polypeptide includes from 3 to 4 lysine amino acids. In embodiments,the polypeptide includes from 4 to 6 lysine amino acids. In embodiments,the polypeptide includes from 4 to 5 lysine amino acids. In embodiments,the polypeptide includes from 5 to 6 lysine amino acids.

In embodiments, the polypeptide includes one (e.g. no more than one)Guanidinium group. In embodiments, the polypeptide includes two (e.g. nomore than two) Guanidinium groups. In embodiments, the polypeptideincludes three (e.g. no more than three) Guanidinium groups. Inembodiments, the polypeptide includes four (e.g. no more than four)Guanidinium groups. In embodiments, the polypeptide includes five (e.g.no more than five) Guanidinium groups. In embodiments, the polypeptideincludes six (e.g. no more than six) Guanidinium groups. In embodiments,the polypeptide includes from 1 to 6 Guanidinium groups. In embodiments,the polypeptide includes from 1 to 5 Guanidinium groups. In embodiments,the polypeptide includes from 1 to 4 Guanidinium groups. In embodiments,the polypeptide includes from 1 to 3 Guanidinium groups. In embodiments,the polypeptide includes from 1 to 2 Guanidinium groups. In embodiments,the polypeptide includes from 2 to 5 Guanidinium groups. In embodiments,the polypeptide includes from 2 to 4 Guanidinium groups. In embodiments,the polypeptide includes from 2 to 3 Guanidinium groups. In embodiments,the polypeptide includes from 3 to 6 Guanidinium groups. In embodiments,the polypeptide includes from 3 to 5 Guanidinium groups. In embodiments,the polypeptide includes from 3 to 4 Guanidinium groups. In embodiments,the polypeptide includes from 4 to 6 Guanidinium groups. In embodiments,the polypeptide includes from 4 to 5 Guanidinium groups. In embodiments,the polypeptide includes from 5 to 6 Guanidinium groups.

In embodiments, the polypeptide includes one (e.g. no more than one)positive charge. In embodiments, the polypeptide includes two (e.g. nomore than two) positive charges. In embodiments, the polypeptideincludes three (e.g. no more than three) positive charges. Inembodiments, the polypeptide includes four (e.g. no more than four)positive charges. In embodiments, the polypeptide includes five (e.g. nomore than five) positive charges. In embodiments, the polypeptideincludes six (e.g. no more than six) positive charges. In embodiments,the polypeptide includes from 1 to 6 positive charges. In embodiments,the polypeptide includes from 1 to 5 positive charges. In embodiments,the polypeptide includes from 1 to 4 positive charges. In embodiments,the polypeptide includes from 1 to 3 positive charges. In embodiments,the polypeptide includes from 1 to 2 positive charges. In embodiments,the polypeptide includes from 2 to 5 positive charges. In embodiments,the polypeptide includes from 2 to 4 positive charges. In embodiments,the polypeptide includes from 2 to 3 positive charges. In embodiments,the polypeptide includes from 3 to 6 positive charges. In embodiments,the polypeptide includes from 3 to 5 positive charges. In embodiments,the polypeptide includes from 3 to 4 positive charges. In embodiments,the polypeptide includes from 4 to 6 positive charges. In embodiments,the polypeptide includes from 4 to 5 positive charges. In embodiments,the polypeptide includes from 5 to 6 positive charges.

In embodiments, the one or more arginine amino acids are at the carboxyterminus of the polypeptide. In embodiments, the one or more lysineamino acids are at the carboxy terminus of the polypeptide. Inembodiments, the one or more guanidinium groups are at the carboxyterminus of the polypeptide. In embodiments, the one or more positivecharges are at the carboxy terminus of the polypeptide. In embodiments,the two or more arginine amino acids are at the carboxy terminus of thepolypeptide. In embodiments, the two or more lysine amino acids are atthe carboxy terminus of the polypeptide. In embodiments, the two or moreguanidinium groups are at the carboxy terminus of the polypeptide. Inembodiments, the two or more positive charges are at the carboxyterminus of the polypeptide. In embodiments wherein one or more aminoacid or charge (e.g., Arg, Lys, guanidinium, positive charge) at at thecarboxy terminus, the one or more amino acids or charges are the aminoacids or units/monomeric units (e.g., positively charged or guanidiniumcontaining units or monomeric units) that are the carboxy terminus (i.e,there are no additional amino acids or units/monomeric units in thepolymer that are carboxy terminal to those amino acids orunits/monomeric units). In embodiments, the one or more arginine aminoacids are one residue from the carboxy terminus of the polypeptide. Inembodiments, the one or more lysine amino acids are one residue from thecarboxy terminus of the polypeptide. In embodiments, the one or moreguanidinium groups are one residue from the carboxy terminus of thepolypeptide. In embodiments, the one or more positive charges are onepolymer unit (residue/amino acid) from the carboxy terminus of thepolypeptide. In embodiments, the two or more arginine amino acids aretogether one residue from the carboxy terminus of the polypeptide. Inembodiments, the two or more lysine amino acids are together one residuefrom the carboxy terminus of the polypeptide. In embodiments, the two ormore guanidinium groups are together one residue or unit from thecarboxy terminus of the polypeptide. In embodiments, the two or morepositive charges are together one unit or residue from the carboxyterminus of the polypeptide.

In embodiments, the one or more arginine amino acids are in the interiorof the polypeptide. In embodiments, the one or more lysine amino acidsare in the interior of the polypeptide. In embodiments, the one or moreguanidinium groups are in the interior of the polypeptide. Inembodiments, the one or more positive charges are in the interior of thepolypeptide. In embodiments, the two or more arginine amino acids are inthe interior of the polypeptide. In embodiments, the two or more lysineamino acids are in the interior of the polypeptide. In embodiments, thetwo or more guanidinium groups are in the interior of the polypeptide.In embodiments, the two or more positive charges are in the interior ofthe polypeptide.

In embodiments, the one or more arginine amino acids are at the aminoterminus of the polypeptide. In embodiments, the one or more lysineamino acids are at the amino terminus of the polypeptide. Inembodiments, the one or more guanidinium groups are at the aminoterminus of the polypeptide. In embodiments, the one or more positivecharges are at the amino terminus of the polypeptide. In embodiments,the two or more arginine amino acids are at the amino terminus of thepolypeptide. In embodiments, the two or more lysine amino acids are atthe amino terminus of the polypeptide. In embodiments, the two or moreguanidinium groups are at the amino terminus of the polypeptide. Inembodiments, the two or more positive charges are at the amino terminusof the polypeptide.

In embodiments, the polypeptide includes from 2 to 6 arginine residues.In embodiments, the polypeptide includes 2 arginine residues. Inembodiments, the arginine residues are the carboxyl terminal residues ofthe polypeptide. In embodiments, the polypeptide does not include acarboxy terminal lysine residue. In embodiments, the polypeptide isresistant to proteolysis relative to the unpolymerized polypeptide.

In embodiments, the polypeptide is resistant to proteolysis. Inembodiments, the polypeptide in the polymer is resistant to proteolysisrelative to the free polypeptide. In embodiments, the polymerpolypeptide is 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% moreresistant to proteolysis than the free polypeptide under the sameconditions. In embodiments, the polymer polypeptide is 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,10000, 100000-fold more stable than the free polypeptide under the sameconditions (e.g., after systemic administration, oral administration,injection). Resistance to proteolysis may be measured by catalyticefficiency, initial rate of proteolysis, kcat/Km (specificity constant),time to proteolyze a fixed amount of polypeptide, degree of proteolysisafter a fixed time, among other well-known methods. In embodiments, thepolymer polypeptide remains intact

In embodiments, the polypeptide (e.g. O; R³ or R⁴) includes amino acidswith alternate backbones from the naturally occurring protein backbone(e.g., peptoids, β-peptides, D-peptides.

In embodiments, the polypeptide includes a peptide tag. In embodiments,the polypeptide includes a protein tag. In embodiments, the polypeptidecontacts (e.g, is bonded to) a ³²P, fluorescent dye, electron-densereagent, enzyme (e.g., as commonly used in an ELISA), biotin,digoxigenin, paramagnetic molecule, paramagnetic nanoparticle,ultrasmall superparamagnetic iron oxide (“USPIO”) nanoparticles, USPIOnanoparticle aggregate, superparamagnetic iron oxide (“SPIO”)nanoparticle, SPIO nanoparticle aggregate, monochrystalline SPIO,monochrystalline SPIO aggregate, monochrystalline iron oxidenanoparticle, monochrystalline iron oxide, other nanoparticle contrastagent, liposome or other delivery vehicle containing Gadolinium chelate(“Gd-chelate”) molecule, Gadolinium, radioisotope, radionuclide (e.g.carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82),fluorodeoxyglucose (e.g. fluorine-18 labeled), any gamma ray emittingradionuclids, positron-emitting radionuclide, radiolabeled glucose,radiolabeled water, radiolabeled ammonia, biocolloids, microbubble (e.g.including microbubble shell including albumin, galactose, lipid, and/orpolymers; microbubble gas core including air, heavy gas(es),perfluorcarbon, nitrogen, octafluoropropane, perflexane lipidmicrosphere, perflutren, etc.), iodinated contrast agent (e.g. iohexol,iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate,metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, goldnanoparticle, gold nanoparticle aggregate, fluorophore, two-photonfluorophore, or hapten. In embodiments, the polypeptide includespoly-histidine (poly(His)), chitin binding protein, maltose bindingprotein, glutathione-S-transferase (GST), FLAG-tag, AviTag,Calmodulin-tag, polyglutamate tag, E-tag, HA-tag, Myc-tag, S-tag,SBP-tag, Softtag 1, Softtag 3, Strep-tag, TC tag, V5 tag, VSV tag,Xpress tag, Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein (BCCP)tag, Halo-tag, thioredoxin-tag, or Fc-tag. In embodiments, the polymerincludes a detectable moiety.

In embodiments, the polypeptide includes a therapeutic polypeptide(e.g., KLA polypeptide). In embodiments, the polypeptide is atherapeutic polypeptide.

In embodiments, the polypeptide includes Nesiritide, Ceruletide,Bentiromide, Exenatide, Gonadorelin, Enfuvirtide, Vancomycin, Icatibant,Secretin, Leuprolide, Glucagon recombinant, Oxytocin, Bivalirudin,Sermorelin, Gramicidin D, Insulin recombinant, Capreomycin, SalmonCalcitonin, Vasopressin, Cosyntropin, Bacitracin, Octreotide, Abarelix,Vapreotide, Thymalfasin, Insulin recombinant, Mecasermin, Cetrorelix,Teriparatide, Corticotropin, or Pramlintide. In embodiments, thepolypeptide is Nesiritide, Ceruletide, Bentiromide, Exenatide,Gonadorelin, Enfuvirtide, Vancomycin, Icatibant, Secretin, Leuprolide,Glucagon recombinant, Oxytocin, Bivalirudin, Sermorelin, Gramicidin D,Insulin recombinant, Capreomycin, Salmon Calcitonin, Vasopressin,Cosyntropin, Bacitracin, Octreotide, Abarelix, Vapreotide, Thymalfasin,Insulin recombinant, Mecasermin, Cetrorelix, Teriparatide,Corticotropin, or Pramlintide. In embodiments, the polypeptide includesTirofiban, captopril, eptifibatide, ziconotide, teriparatide,liraglutide, lanreotide, pramlintide, enfuvirtide, icatibant,ecallantide, tesamorelin, degarelix, mifamurtide, nesiritide, buserelin,gonadorelin, goserelin, histrelin, leuprolide, nafarelin, triptorelin,abarelix, cetrorelix, or ganirelix. In embodiments, the polypeptide isTirofiban, captopril, eptifibatide, ziconotide, teriparatide,liraglutide, lanreotide, pramlintide, enfuvirtide, icatibant,ecallantide, tesamorelin, degarelix, mifamurtide, nesiritide, buserelin,gonadorelin, goserelin, histrelin, leuprolide, nafarelin, triptorelin,abarelix, cetrorelix, or ganirelix.

In embodiments, the polypeptide includes Tat (YGRKKRRQRRR). Inembodiments, the polypeptide includes Arg8 (RRRRRRRR). In embodiments,the polypeptide is Tat (YGRKKRRQRRR). In embodiments, the polypeptide isArg8 (RRRRRRRR).

In embodiments, R¹ includes a solid support (and optionally a covalentlinker to the solid support). In embodiments, R¹ includes a nanoparticle(and optionally a covalent linker to the solid support). In embodiments,R¹ includes a substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl. Inembodiments, R¹ includes a functional moiety. In embodiments, R¹includes a detectable moiety. In embodiments, R¹ includes a peptide tag,protein tag, ³²P, fluorescent dye, electron-dense reagent, enzyme (e.g.,as commonly used in an ELISA), biotin, digoxigenin, paramagneticmolecule, paramagnetic nanoparticle, ultrasmall superparamagnetic ironoxide (“USPIO”) nanoparticles, USPIO nanoparticle aggregate,superparamagnetic iron oxide (“SPIO”) nanoparticle, SPIO nanoparticleaggregate, monochrystalline SPIO, monochrystalline SPIO aggregate,monochrystalline iron oxide nanoparticle, monochrystalline iron oxide,other nanoparticle contrast agent, liposome or other delivery vehiclecontaining Gadolinium chelate (“Gd-chelate”) molecule, Gadolinium,radioisotope, radionuclide (e.g. carbon-11, nitrogen-13, oxygen-15,fluorine-18, rubidium-82), fluorodeoxyglucose (e.g. fluorine-18labeled), any gamma ray emitting radionuclids, positron-emittingradionuclide, radiolabeled glucose, radiolabeled water, radiolabeledammonia, biocolloids, microbubble (e.g. including microbubble shellincluding albumin, galactose, lipid, and/or polymers; microbubble gascore including air, heavy gas(es), perfluorcarbon, nitrogen,octafluoropropane, perflexane lipid microsphere, perflutren, etc.),iodinated contrast agent (e.g. iohexol, iodixanol, ioversol, iopamidol,ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate,thorium dioxide, gold, gold nanoparticle, gold nanoparticle aggregate,fluorophore, two-photon fluorophore, hapten, poly-histidine (poly(His)),chitin binding protein, maltose binding protein,glutathione-S-transferase (GST), FLAG-tag, AviTag, Calmodulin-tag,polyglutamate tag, E-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softtag 1,Softtag 3, Strep-tag, TC tag, V5 tag, VSV tag, Xpress tag, Isopeptag,SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tag, Halo-tag,thioredoxin-tag, or Fc-tag. In embodiments, R¹ includes a polymerizationproduct of an ethyl vinyl ether. In embodiments, R¹ is thepolymerization product of an alkene containing substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl. In embodiments, R¹ is the polymerizationproduct of an alkene bonded to a substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl or substituted or unsubstituted heteroaryl. Inembodiments, R¹ is the polymerization product of an alkene containingcompound (e.g., also including a function group, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, therapeutic moiety, or detectable moiety). Inembodiments, R¹ is selected from:

In embodiments, R² includes a solid support (and optionally a covalentlinker to the solid support). In embodiments, R² includes a nanoparticle(and optionally a covalent linker to the solid support). In embodiments,R² includes a substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl. Inembodiments, R² includes a functional moiety. In embodiments, R²includes a therapeutic moiety. In embodiments, R² includes a detectablemoiety. In embodiments, R² includes a peptide tag, protein tag, ³²P,fluorescent dye, electron-dense reagent, enzyme (e.g., as commonly usedin an ELISA), biotin, digoxigenin, paramagnetic molecule, paramagneticnanoparticle, ultrasmall superparamagnetic iron oxide (“USPIO”)nanoparticles, USPIO nanoparticle aggregate, superparamagnetic ironoxide (“SPIO”) nanoparticle, SPIO nanoparticle aggregate,monochrystalline SPIO, monochrystalline SPIO aggregate, monochrystallineiron oxide nanoparticle, monochrystalline iron oxide, other nanoparticlecontrast agent, liposome or other delivery vehicle containing Gadoliniumchelate (“Gd-chelate”) molecule, Gadolinium, radioisotope, radionuclide(e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82),fluorodeoxyglucose (e.g. fluorine-18 labeled), any gamma ray emittingradionuclids, positron-emitting radionuclide, radiolabeled glucose,radiolabeled water, radiolabeled ammonia, biocolloids, microbubble (e.g.including microbubble shell including albumin, galactose, lipid, and/orpolymers; microbubble gas core including air, heavy gas(es),perfluorcarbon, nitrogen, octafluoropropane, perflexane lipidmicrosphere, perflutren, etc.), iodinated contrast agent (e.g. iohexol,iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate,metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, goldnanoparticle, gold nanoparticle aggregate, fluorophore, two-photonfluorophore, hapten, poly-histidine (poly(His)), chitin binding protein,maltose binding protein, glutathione-S-transferase (GST), FLAG-tag,AviTag, Calmodulin-tag, polyglutamate tag, E-tag, HA-tag, Myc-tag,S-tag, SBP-tag, Softtag 1, Softtag 3, Strep-tag, TC tag, V5 tag, VSVtag, Xpress tag, Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein(BCCP) tag, Halo-tag, thioredoxin-tag, or Fc-tag. In embodiments, R²includes a polymerization product of an ethyl vinyl ether. Inembodiments, R² is the polymerization product of an alkene containingsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl orsubstituted or unsubstituted heteroaryl. In embodiments, R² is thepolymerization product of an alkene bonded to a substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl. In embodiments, R² is the polymerizationproduct of an alkene containing compound (e.g., also including afunction group, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, therapeuticmoiety, or detectable moiety). In embodiments, R² is selected from:

In embodiments, R¹ includes a substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl or substituted or unsubstituted heteroaryl. Inembodiments, R² includes a substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl or substituted or unsubstituted heteroaryl.

A “non-polypeptide monomer” is a polymerizable monomer or polymerizedmonomer that does not include a polypeptide. A “non-polypeptide moiety”is the moiety covalently attached to a polymerizable monomer orpolymerized monomer that does not include a polypeptide. Anon-polypeptide monomer may be a polymerizable monomer or polymerizedmonomer covalently bound to a substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, a non-polypeptide monomer or polymerized monomer is ahydrophobic monomer. In embodiments, each non-polypeptide monomer orpolymerized monomer in the polymer is optionally different. Inembodiments, each non-polypeptide monomer (polymerized monomer) in thepolymer is identical. In embodiments, the polymer includes blocks ofnon-polypeptide monomers (polymerized monomers) wherein thenon-polypeptide moiety in each block are identical and thenon-polypeptide moiety in different blocks are optionally different. Inembodiments, the polymer includes blocks of non-polypeptide monomers(polymerized monomers) wherein the non-polypeptide moieties in eachblock are identical and the non-polypeptide moieties in different blocksare different. In embodiments, the non-polypeptide polymerizable monomeris selected from:

The above referenced non-polypeptide polymerizable monomer formpolymerized monomers upon incorporation into the polymers describedherein. In embodiments, a non-polypeptide moiety is a hydrophobicmoiety. In embodiments, a non-polypeptide moiety does not include anoligonucleotide. In embodiments, a non-polypeptide moiety does notinclude a nucleotide. In embodiments, a non-polypeptide moiety does notinclude a nucleobase (e.g., A, T, C, G, U). In embodiments, thenon-polypeptide moiety is a substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl andwherein said non-polypeptide moiety does not comprise a nucleotide. Inembodiments, the non-polypeptide moiety is a hydrophobic moiety. Inembodiments, the non-polypeptide moiety does not include anoligonucleotide. In embodiments, the non-polypeptide moiety is asubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In embodiments, thenon-polypeptide moiety does not include a nucleotide. In embodiments,the non-polypeptide moiety does not include a polypeptide. Inembodiments, the non-polypeptide moiety does not include an amino acid.In embodiments, the non-polypeptide moiety does not include anoligonucleotide. In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, the hydrophobic moiety is sufficiently hydrophobic andof sufficient size such that the block polymer is capable of forming amicelle in an aqueous-based solvent. In embodiments, the hydrophobicmoiety is a substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, the hydrophobic moiety is an unsubstituted benzyl.

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, M(P) is

In embodiments, each hydrophobic moiety in the copolymer is optionallydifferent. In embodiments, each hydrophobic moiety in the copolymer isidentical. In embodiments, the copolymer includes blocks of hydrophobicmoieties wherein the hydrophobic moieties in each block are identicaland the hydrophobic moieties in different blocks are optionallydifferent. In embodiments, the copolymer includes blocks of hydrophobicmoieties wherein the hydrophobic moieties in each block are identicaland the hydrophobic moieties in different blocks are different.

In embodiments, m is an integer from 2 to 900. In embodiments, m is aninteger from 2 to 800. In embodiments, m is an integer from 2 to 700. Inembodiments, m is an integer from 2 to 600. In embodiments, m is aninteger from 2 to 500. In embodiments, m is an integer from 2 to 400. Inembodiments, m is an integer from 2 to 300. In embodiments, m is aninteger from 2 to 200. In embodiments, m is an integer from 2 to 100. Inembodiments, m is an integer from 2 to 50. In embodiments, m is aninteger from 2 to 49. In embodiments, m is an integer from 2 to 48. Inembodiments, m is an integer from 2 to 47. In embodiments, m is aninteger from 2 to 46. In embodiments, m is an integer from 2 to 45. Inembodiments, m is an integer from 2 to 44. In embodiments, m is aninteger from 2 to 43. In embodiments, m is an integer from 2 to 42. Inembodiments, m is an integer from 2 to 41. In embodiments, m is aninteger from 2 to 40. In embodiments, m is an integer from 2 to 39. Inembodiments, m is an integer from 2 to 38. In embodiments, m is aninteger from 2 to 37. In embodiments, m is an integer from 2 to 36. Inembodiments, m is an integer from 2 to 35. In embodiments, m is aninteger from 2 to 34. In embodiments, m is an integer from 2 to 33. Inembodiments, m is an integer from 2 to 32. In embodiments, m is aninteger from 2 to 31. In embodiments, m is an integer from 2 to 30. Inembodiments, m is an integer from 2 to 29. In embodiments, m is aninteger from 2 to 28. In embodiments, m is an integer from 2 to 27. Inembodiments, m is an integer from 2 to 26. In embodiments, m is aninteger from 2 to 25. In embodiments, m is an integer from 2 to 24. Inembodiments, m is an integer from 2 to 23. In embodiments, m is aninteger from 2 to 22. In embodiments, m is an integer from 2 to 21. Inembodiments, m is an integer from 2 to 20. In embodiments, m is aninteger from 2 to 19. In embodiments, m is an integer from 2 to 18. Inembodiments, m is an integer from 2 to 17. In embodiments, m is aninteger from 2 to 16. In embodiments, m is an integer from 2 to 15. Inembodiments, m is an integer from 2 to 14. In embodiments, m is aninteger from 2 to 13. In embodiments, m is an integer from 2 to 12. Inembodiments, m is an integer from 2 to 11. In embodiments, m is aninteger from 2 to 10. In embodiments, m is an integer from 2 to 9. Inembodiments, m is an integer from 2 to 8. In embodiments, m is aninteger from 2 to 7. In embodiments, m is an integer from 2 to 6. Inembodiments, m is an integer from 2 to 5. In embodiments, m is aninteger from 2 to 4. In embodiments, m is an integer from 2 to 3. Inembodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510,511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524,525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538,539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552,553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580,581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608,609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636,637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650,651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664,665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678,679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692,693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706,707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720,721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734,735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748,749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762,763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776,777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790,791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804,805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818,819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832,833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846,847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860,861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874,875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888,889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902,903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916,917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930,931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944,945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958,959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972,973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986,987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or1000.

In embodiments of the blend copolymer described herein, the plurality ofpolypeptide branches and plurality of non-polypeptide side chains may berandomly mixed together through the length of the polymer. Inembodiments, the plurality of polypeptide branches and plurality ofnon-polypeptide side chains are mixed in a repetitive pattern. Forexample, the polymer may include a repeated pattern of n polypeptidemonomers followed by m non-polypeptide monomers, with this patternrepeated z times.

The linear backbone, polypeptides (e.g. O; R³ or R⁴), polypeptidemoieties, non-polypeptide moieties, graft-through polymerization, andpolymerizable or polymerized monomer, are as described herein, includingin aspects (e.g., above), embodiments (e.g., above), examples, figures,tables, schemes, and claims.

In embodiments, z is an integer from 2 to 2000. In embodiments, z is aninteger from 2 to 1000. In embodiments, z is an integer from 2 to 900.In embodiments, z is an integer from 2 to 800. In embodiments, z is aninteger from 2 to 700. In embodiments, z is an integer from 2 to 600. Inembodiments, z is an integer from 2 to 500. In embodiments, z is aninteger from 2 to 400. In embodiments, z is an integer from 2 to 300. Inembodiments, z is an integer from 2 to 200. In embodiments, z is aninteger from 2 to 100. In embodiments, z is an integer from 2 to 50. Inembodiments, z is an integer from 2 to 49. In embodiments, z is aninteger from 2 to 48. In embodiments, z is an integer from 2 to 47. Inembodiments, z is an integer from 2 to 46. In embodiments, z is aninteger from 2 to 45. In embodiments, z is an integer from 2 to 44. Inembodiments, z is an integer from 2 to 43. In embodiments, z is aninteger from 2 to 42. In embodiments, z is an integer from 2 to 41. Inembodiments, z is an integer from 2 to 40. In embodiments, z is aninteger from 2 to 39. In embodiments, z is an integer from 2 to 38. Inembodiments, z is an integer from 2 to 37. In embodiments, z is aninteger from 2 to 36. In embodiments, z is an integer from 2 to 35. Inembodiments, z is an integer from 2 to 34. In embodiments, z is aninteger from 2 to 33. In embodiments, z is an integer from 2 to 32. Inembodiments, z is an integer from 2 to 31. In embodiments, z is aninteger from 2 to 30. In embodiments, z is an integer from 2 to 29. Inembodiments, z is an integer from 2 to 28. In embodiments, z is aninteger from 2 to 27. In embodiments, z is an integer from 2 to 26. Inembodiments, z is an integer from 2 to 25. In embodiments, z is aninteger from 2 to 24. In embodiments, z is an integer from 2 to 23. Inembodiments, z is an integer from 2 to 22. In embodiments, z is aninteger from 2 to 21. In embodiments, z is an integer from 2 to 20. Inembodiments, z is an integer from 2 to 19. In embodiments, z is aninteger from 2 to 18. In embodiments, z is an integer from 2 to 17. Inembodiments, z is an integer from 2 to 16. In embodiments, z is aninteger from 2 to 15. In embodiments, z is an integer from 2 to 14. Inembodiments, z is an integer from 2 to 13. In embodiments, z is aninteger from 2 to 12. In embodiments, z is an integer from 2 to 11. Inembodiments, z is an integer from 2 to 10. In embodiments, z is aninteger from 2 to 9. In embodiments, z is an integer from 2 to 8. Inembodiments, z is an integer from 2 to 7. In embodiments, z is aninteger from 2 to 6. In embodiments, z is an integer from 2 to 5. Inembodiments, z is an integer from 2 to 4. In embodiments, z is aninteger from 2 to 3. In embodiments, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506,507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520,521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534,535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548,549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562,563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576,577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590,591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604,605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618,619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632,633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646,647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660,661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674,675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688,689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716,717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730,731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744,745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758,759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772,773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786,787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800,801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814,815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828,829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842,843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856,857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870,871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884,885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898,899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912,913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926,927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940,941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954,955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968,969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982,983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996,997, 998, 999, or 1000.

In embodiments of the blend copolymer, n is an integer from 2 to 100. Inembodiments, n is an integer from 2 to 50. In embodiments, n is aninteger from 2 to 49. In embodiments, n is an integer from 2 to 48. Inembodiments, n is an integer from 2 to 47. In embodiments, n is aninteger from 2 to 46. In embodiments, n is an integer from 2 to 45. Inembodiments, n is an integer from 2 to 44. In embodiments, n is aninteger from 2 to 43. In embodiments, n is an integer from 2 to 42. Inembodiments, n is an integer from 2 to 41. In embodiments, n is aninteger from 2 to 40. In embodiments, n is an integer from 2 to 39. Inembodiments, n is an integer from 2 to 38. In embodiments, n is aninteger from 2 to 37. In embodiments, n is an integer from 2 to 36. Inembodiments, n is an integer from 2 to 35. In embodiments, n is aninteger from 2 to 34. In embodiments, n is an integer from 2 to 33. Inembodiments, n is an integer from 2 to 32. In embodiments, n is aninteger from 2 to 31. In embodiments, n is an integer from 2 to 30. Inembodiments, n is an integer from 2 to 29. In embodiments, n is aninteger from 2 to 28. In embodiments, n is an integer from 2 to 27. Inembodiments, n is an integer from 2 to 26. In embodiments, n is aninteger from 2 to 25. In embodiments, n is an integer from 2 to 24. Inembodiments, n is an integer from 2 to 23. In embodiments, n is aninteger from 2 to 22. In embodiments, n is an integer from 2 to 21. Inembodiments, n is an integer from 2 to 20. In embodiments, n is aninteger from 2 to 19. In embodiments, n is an integer from 2 to 18. Inembodiments, n is an integer from 2 to 17. In embodiments, n is aninteger from 2 to 16. In embodiments, n is an integer from 2 to 15. Inembodiments, n is an integer from 2 to 14. In embodiments, n is aninteger from 2 to 13. In embodiments, n is an integer from 2 to 12. Inembodiments, n is an integer from 2 to 11. In embodiments, n is aninteger from 2 to 10. In embodiments, n is an integer from 2 to 9. Inembodiments, n is an integer from 2 to 8. In embodiments, n is aninteger from 2 to 7. In embodiments, n is an integer from 2 to 6. Inembodiments, n is an integer from 2 to 5. In embodiments, n is aninteger from 2 to 4. In embodiments, n is an integer from 2 to 3. Inembodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200.

In embodiments of the blend copolymer, m is an integer from 2 to 100. Inembodiments, m is an integer from 2 to 50. In embodiments, m is aninteger from 2 to 49. In embodiments, m is an integer from 2 to 48. Inembodiments, m is an integer from 2 to 47. In embodiments, m is aninteger from 2 to 46. In embodiments, m is an integer from 2 to 45. Inembodiments, m is an integer from 2 to 44. In embodiments, m is aninteger from 2 to 43. In embodiments, m is an integer from 2 to 42. Inembodiments, m is an integer from 2 to 41. In embodiments, m is aninteger from 2 to 40. In embodiments, m is an integer from 2 to 39. Inembodiments, m is an integer from 2 to 38. In embodiments, m is aninteger from 2 to 37. In embodiments, m is an integer from 2 to 36. Inembodiments, m is an integer from 2 to 35. In embodiments, m is aninteger from 2 to 34. In embodiments, m is an integer from 2 to 33. Inembodiments, m is an integer from 2 to 32. In embodiments, m is aninteger from 2 to 31. In embodiments, m is an integer from 2 to 30. Inembodiments, m is an integer from 2 to 29. In embodiments, m is aninteger from 2 to 28. In embodiments, m is an integer from 2 to 27. Inembodiments, m is an integer from 2 to 26. In embodiments, m is aninteger from 2 to 25. In embodiments, m is an integer from 2 to 24. Inembodiments, m is an integer from 2 to 23. In embodiments, m is aninteger from 2 to 22. In embodiments, m is an integer from 2 to 21. Inembodiments, m is an integer from 2 to 20. In embodiments, m is aninteger from 2 to 19. In embodiments, m is an integer from 2 to 18. Inembodiments, m is an integer from 2 to 17. In embodiments, m is aninteger from 2 to 16. In embodiments, m is an integer from 2 to 15. Inembodiments, m is an integer from 2 to 14. In embodiments, m is aninteger from 2 to 13. In embodiments, m is an integer from 2 to 12. Inembodiments, m is an integer from 2 to 11. In embodiments, m is aninteger from 2 to 10. In embodiments, m is an integer from 2 to 9. Inembodiments, m is an integer from 2 to 8. In embodiments, m is aninteger from 2 to 7. In embodiments, m is an integer from 2 to 6. Inembodiments, m is an integer from 2 to 5. In embodiments, m is aninteger from 2 to 4. In embodiments, m is an integer from 2 to 3. Inembodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200.

In embodiments, the polymer may be a block copolymer wherein each blockis a blend copolymer.

In embodiments, the ratio of polypeptide monomers to non-polypeptidemonomers in the polymer is about 1:10000, 1:1000, 1:100, 1:10, 1:1,10:1, 100:1, 1000:1, or 10000:1. In embodiments, the ratio ofpolypeptide monomers to non-polypeptide monomers in the polymer is1:10000, 1:1000, 1:100, 1:10, 1:1, 10:1, 100:1, 1000:1, or 10000:1.

In an aspect is provided a micelle including a polymer (e.g., copolymer)described herein.

In embodiments, the micelle has a longest dimension (e.g. diameter) ofbetween about 1 and about 1000 nm. In embodiments, the micelle has alongest dimension (e.g. diameter) of between about 5 and about 100 nm.In embodiments, the micelle has a longest dimension (e.g. diameter) ofbetween about 10 and about 50 nm. In embodiments, the micelle has alongest dimension (e.g. diameter) of between 1 and 1000 nm. Inembodiments, the micelle has a longest dimension (e.g. diameter) ofbetween 5 and 100 nm. In embodiments, the micelle has a longestdimension (e.g. diameter) of between 10 and 50 nm.

In embodiments, the micelle has a longest dimension (e.g. diameter) ofabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457,458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499,500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513,514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527,528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541,542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555,556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569,570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583,584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597,598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625,626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639,640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653,654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667,668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681,682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695,696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709,710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723,724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737,738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751,752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765,766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779,780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793,794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807,808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821,822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835,836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849,850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863,864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877,878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891,892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905,906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919,920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933,934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947,948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961,962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975,976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989,990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 nm. Inembodiments, the micelle has a diameter of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211,212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337,338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379,380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393,394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421,422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435,436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477,478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519,520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533,534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547,548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561,562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575,576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589,590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603,604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617,618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631,632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645,646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659,660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673,674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687,688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701,702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715,716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729,730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743,744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757,758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771,772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785,786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799,800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813,814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827,828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841,842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855,856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869,870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883,884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897,898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911,912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925,926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939,940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953,954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967,968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981,982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995,996, 997, 998, 999, or 1000 nm.

In embodiments, the micelle longest dimension (e.g. diameter) is ahydrodynamic longest dimension (e.g. diameter). In embodiments, thelongest dimension (e.g. diameter) is an average longest dimension (e.g.diameter) of a sample.

In an aspect is provided a nanoparticle including a polymer (e.g.,copolymer) described herein.

In embodiments, the nanoparticle is a approximately sphericalnanoparticle.

In embodiments, the nanoparticle has a longest dimension (e.g. diameter)of between about 1 and about 1000 nm. In embodiments, the nanoparticlehas a longest dimension (e.g. diameter) of between about 5 and about 100nm. In embodiments, the nanoparticle has a longest dimension (e.g.diameter) of between about 10 and about 50 nm. In embodiments, thenanoparticle has a longest dimension (e.g. diameter) of between 1 and1000 nm. In embodiments, the nanoparticle has a longest dimension (e.g.diameter) of between 5 and 100 nm. In embodiments, the nanoparticle hasa longest dimension (e.g. diameter) of between 10 and 50 nm.

In embodiments, the nanoparticle has a longest dimension (e.g. diameter)of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316,317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344,345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400,401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456,457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470,471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498,499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512,513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526,527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540,541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568,569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582,583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596,597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610,611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638,639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652,653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666,667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680,681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694,695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708,709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722,723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736,737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750,751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764,765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778,779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792,793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806,807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820,821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834,835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848,849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862,863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876,877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890,891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904,905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918,919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932,933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946,947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960,961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974,975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988,989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 nm.

In embodiments, the nanoparticle has a longest dimension (e.g. diameter)of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457,458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499,500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513,514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527,528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541,542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555,556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569,570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583,584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597,598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625,626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639,640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653,654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667,668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681,682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695,696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709,710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723,724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737,738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751,752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765,766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779,780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793,794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807,808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821,822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835,836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849,850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863,864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877,878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891,892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905,906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919,920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933,934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947,948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961,962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975,976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989,990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 nm.

In embodiments, the nanoparticle longest dimension (e.g. diameter) is ahydrodynamic longest dimension (e.g. diameter). In embodiments, thelongest dimension (e.g. diameter) is an average longest dimension (e.g.diameter) of a sample.

In embodiments, the polymer is capable of programmed self-assembly. Inembodiments, the programmed self-assembly results in a nanoparticle(e.g., as described herein).

In embodiments, the polymer is a capable of cellular internalization.

In an aspect is provided a polymer including a linear backbonecovalently bound to a plurality of polypeptide branches. The polymer(graft polymer) is assembled by graft-through polymerization of aplurality of polypeptide monomers including a polymerizable monomercovalently bound to an polypeptide. The polypeptide thereby forming eachof the plurality of polypeptide branches.

In embodiments, the graft-through polymerization employs ring-openingmetathesis polymerization (ROMP). In embodiments, the graft-throughpolymerization includes ring-opening metathesis polymerization (ROMP).In embodiments, the graft-through polymerization employs radicalpolymerization, controlled radical polymerization, reversibleaddition-fragmentation chain transfer (RAFT) polymerization, atomtransfer radical polymerization (ATRP), ring-opening metathesispolymerization (ROMP), anionic polymerization, cationic polymerization,free radical living polymerization, acyclic diene metathesispolymerization, radiation-induced polymerization, ring-opening olefinmetathesis polymerization, polycondensation reactions, oriniferter-induced polymerization.

In an aspect is provided a block copolymer including a linear backbonecovalently bound to a plurality of polypeptide (e.g. O; R³ or R⁴) moietybranches and a plurality of non-polypeptide moiety side chains, wherein:the plurality of polypeptide moiety branches form a first block portionof the copolymer and the non-polypeptide moiety side chains form asecond block portion of the copolymer; the copolymer is assembled bygraft-through polymerization of a plurality of polypeptide moietymonomers and a plurality of non-polypeptide moiety monomers, whereineach of the plurality of polypeptide moiety monomers includes apolymerizable monomer covalently bound to an polypeptide moiety, thepolypeptide moiety thereby forming each of the plurality of polypeptidemoiety branches; and each of the plurality of non-polypeptide moietymonomers includes the polymerizable monomer covalently bound to anon-polypeptide moiety, the non-polypeptide moiety thereby forming eachof the plurality of non-polypeptide moiety side chains.

In an aspect is provided an amphiphilic block copolymer including alinear backbone covalently bound to a plurality of polypeptide (e.g. O;R³ or R⁴) moiety branches and a plurality of hydrophobic moiety sidechains, wherein: the plurality of polypeptide moiety branches form ahydrophilic block portion of the amphiphilic copolymer and thehydrophobic moiety side chains form a hydrophobic block portion of theamphiphilic copolymer; the copolymer is assembled by graft-throughpolymerization of a plurality of polypeptide moiety monomers and aplurality of hydrophobic moiety monomers, wherein each of the pluralityof polypeptide moiety monomers includes a polymerizable monomercovalently bound to a polypeptide moiety, the polypeptide moiety therebyforming each of the plurality of polypeptide moiety branches; and eachof the plurality of hydrophobic moiety monomers includes thepolymerizable monomer covalently bound to a hydrophobic moiety, thehydrophobic moiety thereby forming each of the plurality of hydrophobicmoiety side chains.

The linear backbone, polypeptide (e.g. O; R³ or R⁴) moiety branches,polypeptide monomers, graft-through polymerization, polymerizablemonomer, and polypeptide are as described herein, including in aspects(e.g., above), embodiments (e.g., above), examples, figures, tables,schemes, and claims.

In embodiments, the amphiphilic block copolymer has the formula:R¹-[M(O)]_(n)-[M(P)]_(m)—R² or R¹-[M(P)]_(m)-[M(O)]_(n)—R² wherein, n isan integer from 2 to 1000; m is an integer from 2 to 1000; M is thepolymerized monomer; O is independently apolypeptide; P is independentlyahydrophobic moiety; and R¹ and R² are independently terminal polymermoieties.

R¹, M, O, n, and R² are as described herein, including in aspects (e.g.,above), embodiments (e.g., above), examples, figures, tables, schemes,and claims.

In an aspect is provided a micelle including an amphiphilic blockcopolymer described herein, including in an aspect, embodiment, example,figures, table, scheme, or claim.

In an aspect is provided a nanoparticle including an amphiphilic blockcopolymer described herein, including in an aspect, embodiment, example,figure, table, scheme, or claim.

In an aspect is provided a blend copolymer including a linear backbonecovalently bound to a plurality of polypeptide (e.g. O; R³ or R⁴) moietybranches and a plurality of non-polypeptide moiety side chains, whereinthe plurality of polypeptide moiety branches and plurality ofnon-polypeptide moiety side chains are mixed.

In embodiments, the polymer, micelle, amphiphilic block copolymer, blendcopolymer, or nanoparticle is as described herein, including in anaspect, embodiment, example, figure, table, scheme, and claim.

In one aspect the present disclosure provides a cell penetrating highdensity brush peptide polymer composition and/or delivery vehicle fordelivering cell penetrating high density brush peptide polymer anddrugs. In the composition and/or the delivery vehicle of the presentdisclosure the peptide is active on the polymer or may be released at agiven place and time via a cleavable linkage.

In one aspect the present disclosure provides a composition including ahigh density brush peptide polymer, where the peptide is pendant topolymerized norbornene moieties. In one aspect, the present disclosureincludes a drug delivery vehicle including a high density brush peptidepolymer, where the peptide is pendant to polymerized norbornene moieties(polymerized moieties).

The norbornene may be a cis-5-norbornene-exo-2,3-dicarboxylic anhydride.In embodiments, the norbornene moieties are N-(hexanoicacid)-cis-5-norbornene-exo-dicarboximide (NorAha) moieties orN-(glycine)-cis-5-norbornene-exo-dicarboximide (NorGly) moieties.

In embodiments, blend co-polymers are prepared with in various ratios ofblock polymers. For example, 9:1 ratio blend co-polymer with the peptidemonomer (m=1) at the end or in the middle of the polymer can beprepared, or an intermediate 5:5 ratio blend co-polymer can be prepared,shown below.

In embodiments, the present disclosure includes the high density brushpeptide homopolymer or a block copolymer. In embodiments, the degree ofpolymerization of the homopolymer may be 8-70. In embodiments, thedegree of polymerization of the homopolymer is 8, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, or any intervening digit.

In embodiments, the non-peptidic moieties is oligoethylene glycol (OEG),poly(ethylene glycol) 5000 (PEG 5000), or poly(ethylene glycol) 2000(PEG 2000).

In embodiments, the norbornene moieties are linked to the polypeptide ornon-polypeptide moiety with a linker. In embodiments, the linker is acarbon linker. In embodiments, the linker is a substituted orunsubstituted C₁-C₂₀ alkylene. The linker is a carboxylic acid of asubstituted or unsubstituted C₁-C₂₀ alkylene. In embodiments, the linkeris hexanoic acid of methane, ethane, propane, butane, pentane, hexane,heptane, octane, nonane, or decane.

In embodiments, the polypetide moiety includes peptides with arginine orlysine residues at the N- or at the C-terminus. In embodiments, thepolymer includes peptide with arginine or lysine residues at theC-terminus. In embodiments, the polymer includes peptide with one or twoarginine or lysine residues at the C-terminus. In embodiments, thepolymer includes peptide with two arginine residues at the C-terminus.The C-terminus faces the solution. In embodiments, the polymer includespeptides with two arginine residues at the C-terminus. In embodiments,the polymer includes a therapeutic polypeptide. Non-limiting examples ofthe therapeutic polypeptide includes: a cell growth or proliferationinhibitory peptide, an anti-inflammatory peptide, an anti-tumor oranti-cancer peptide, an anti-apoptotic peptide, anti-diabetic,anti-obesity, anti-infective, anti-bacterial, anti-viral, peptides forpromoting cell growth and differentiation, peptides for preventing pain,and peptides for preventing or treating neural degeneration and/orpeptides for promoting neurogenesis.

In embodiments, the present disclosure includes a cell penetrating highdensity brush peptide polymer composition including: Nesiritide,Ceruletide, Bentiromide, Exenatide, Gonadorelin, Enfuvirtide,Vancomycin, Icatibant, Secretin, Leuprolide, Glucagon recombinant,Oxytocin, Bivalirudin, Sermorelin, Gramicidin D, Insulin recombinant,Capreomycin, Salmon Calcitonin, Vasopressin, Cosyntropin, Bacitracin,Octreotide, Abarelix, Vapreotide, Thymalfasin, Insulin recombinant,Mecasermin, Cetrorelix, Teriparatide, Corticotropin, or Pramlintide.

In embodiments, the peptide in the polymer is resistant to proteolyticdegradation.

In embodiments, the high density brush peptide polymer forms sphericalmicelles of about 10-50 nm diameter (rephrase). In embodiments, a sidechain protecting group—Pbf—is attached to the peptide during peptidesynthesis.

In embodiments, the present disclosure includes norbornene monomers andpolymerized high density peptide polymers prepared by ROMP. The highdensity peptide brush polymers of the present disclosure are lesssusceptible to proteolytic degradation compared to linear peptidesequences. In embodiments, the high density brush polymers are resistantto proteolysis and efficiently penetrate and carry a cargo into thecell.

In embodiments, the peptide brush polymers of the present disclosureforms ˜10-50 nm diameter spherical micellar assemblies and penetratescells efficiently. In embodiments, both polymer and particleformulations of the peptide brush polymers are cell penetrating peptidesthat are resistant to proteolysis under conditions that readily degradethe non-polymerized

In embodiments, the polymer includes a longer linker between thenorbornyl polymer backbone and the peptide in order to separate thepeptide from the polymer backbone, thereby making the peptides moreflexible and sterically accessible. In embodiments, the linkers arecleavable under appropriate biological conditions or by exogenoussources. Appropriate pH- or UV-sensitive linkers may be included in thepolymers. Polymer-drug conjugates improve drug solubility, circulationtime (through the properties of the polymer carrier), and drug targeting(via the use of appropriate linkers that can respond to changes inphysiological conditions such as temperature, pH, and the presence ofenzymes). In this type of polymer conjugate, multiple copies ofbioactive agents, ranging from small molecule drugs to larger compoundslike

Pharmaceutical Compositions

In another aspect is provided a pharmaceutical composition including apharmaceutically acceptable excipient and a polymer (e.g., polypeptidepolymer, copolymer, block copolymer, blend copolymer), micelle, ornanoparticle (e.g, each as described herein) as described herein.

In embodiments of the pharmaceutical compositions, the polymer (e.g.,polypeptide polymer, copolymer, block copolymer, blend copolymer),micelle, or nanoparticle (e.g, each as described herein), is included ina therapeutically effective amount.

In embodiments of the pharmaceutical compositions, the pharmaceuticalcomposition includes a second polymer (e.g., polypeptide polymer,copolymer, block copolymer, blend copolymer), micelle, or nanoparticle(e.g, each as described herein). In embodiments of the pharmaceuticalcompositions, the pharmaceutical composition includes a second polymer(e.g., polypeptide polymer, copolymer, block copolymer, blendcopolymer), micelle, or nanoparticle (e.g, each as described herein) ina therapeutically effective amount.

Methods of Using Polymers

In an aspect is provided a method of administering a polypeptide to theinterior of a cell including contacting the cell with a polymer (e.g.,polypeptide polymer, copolymer, block copolymer, blend copolymer),micelle, or nanoparticle (e.g, each as described herein).

In embodiments, the cell is in a subject. In embodiments, the polymer,micelle, or nanoparticle is administered systemically to the subject.

In an aspect is provided a method of internalizing polypeptides (e.g.,polypeptides copolymers, block copolymers, blend copolymers) into a cellincluding contacting the cell with a polymer. In embodiments, thepolymer is described herein, including in an aspect, embodiment,example, figure, table, scheme, or claim. In embodiments, thepolypeptide is a polypeptide as described herein.

In embodiments, the polymer is a polymer described herein (including inan aspect, embodiment, example, figure, table, claim, or scheme). Inembodiments, the polymer is a brush polymer. In embodiments, the polymeris a block copolymer. In embodiments, the polymer is a block copolymerdescribed herein, including in an aspect, embodiment, example, figure,table, scheme, or claim. In embodiments, the polymer is a blendcopolymer (e.g., described herein).

In one aspect, the present disclosure provides a method of delivering apeptide, by the delivery device of the present disclosure.

Methods of Treatment

In an aspect is provided a method of treating a disease in a subject,including administering to the subject an effective amount of a polymerdescribed herein (e.g., polypeptide polymer, copolymer, block copolymer,blend copolymer), micelle, or nanoparticle (e.g., each as describedherein).

In embodiments, the subject is human. In embodiments, the polymer,micelle, or nanoparticle is administered systemically to the subject.

In an aspect is provided a method of internalizing polypeptides (e.g.,polypeptides copolymers, block copolymers, blend copolymers) into a cellincluding contacting the cell with a polymer. In embodiments, thepolymer is described herein, including in an aspect, embodiment,example, figure, table, scheme, or claim. In embodiments, thepolypeptide is a polypeptide as described herein.

In embodiments, the polymer is a polymer described herein (including inan aspect,

ADDITIONAL EMBODIMENTS

1a. A composition comprising a cell penetrating high density brushpeptide polymer, wherein the peptide polymer comprises peptides witharginine or lysine residues appended at the N- or at the C-terminus, andthe peptide is pendant to polymerized norbornene moieties.2a. A drug delivery vehicle for delivering a therapeutic agent into acell, comprising a cell penetrating high density brush peptide polymer,wherein the peptide polymer comprises peptides with arginine or lysineresidues appended at the N- or at the C-terminus, and peptide is pendantto polymerized norbornene moieties.3a. The composition of any previous embodiment or the drug deliveryvehicle of any previous embodiment, wherein the high density brushpeptide polymer is linked to a small molecule pharmaceutically activeagent or an oligonucleotide4a. The composition or the drug delivery vehicle of one of aboveembodiments, wherein the polymer comprises peptide with arginine orlysine residues at the C-terminus.5a. The composition or the drug delivery vehicle of one of aboveembodiments, wherein the polymer comprises peptide with one or twoarginine or lysine residues at the C-terminus.6a. The composition or the drug delivery vehicle of one of aboveembodiments, wherein the polymer comprises peptide with two arginineresidues at the C-terminus.7a. The composition of any previous embodiment or the drug deliveryvehicle of any previous embodiment, wherein the high density brushpeptide polymer is a homopolymer or a block copolymer.8a. The composition or the drug delivery vehicle of any previousembodiment, wherein the degree of polymerization of the homopolymer is8-70.9a. The composition or the drug delivery vehicle of any previousembodiment, wherein the degree of polymerization of the homopolymer is8, 15, 30, or 60.10a. The composition or the drug delivery vehicle of any previousembodiment, wherein the block copolymer comprises a first block polymercomprising a peptide pendant to polymerized norbornene moieties, and asecond block polymer comprising a peptidic or non-peptidic moietypendant to norbornene moieties, wherein the degree of polymerization ofthe first block is 8-70, and the degree of polymerization of the secondblock is 10-30.11a. The composition or the drug delivery vehicle of one of aboveembodiments, wherein the norbornene moieties are linked to the peptideor the non-peptidic moiety with a linker.12a. The composition or the drug delivery vehicle of any previousembodiment, wherein the linker is a carbon linker.13a. The composition or the drug delivery vehicle any previousembodiment, wherein the carbon linker is a substituted or unsubstitutedC₁-C₂₀ alkane.14a. The composition or the drug delivery vehicle of any previousembodiment, wherein the linker comprises a carboxylic acid of asubstituted or unsubstituted C₁-C₂₀ alkane.15a. The composition or the drug delivery vehicle of any previousembodiment wherein the linker is hexanoic acid of methane, ethane,propane, butane, pentane, hexane, heptane, octane, nonane, or decane.16a. The composition or the drug delivery vehicle of one of aboveembodiments, wherein the norbornene moieties are5-norbornene-exo-dicarboximide moieties.17a. The composition or the drug delivery vehicle of embodiment 18,wherein the norbornene moieties are N-(hexanoicacid)-cis-5-norbornene-exo-dicarboximide (NorAha) moieties orN-(glycine)-cis-5-norbornene-exo-dicarboximide (NorGly) moieties.18a. The composition or the drug delivery vehicle of embodiment 11,wherein the non-peptidic moiety is oligoethylene glycol (OEG),poly(ethylene glycol) 5000 (PEG 5000), or poly(ethylene glycol) 2000(PEG 2000).29a. The composition or the drug delivery vehicle of one of aboveembodiments, wherein the polymer comprises a therapeutic peptide.20a. The composition or the drug delivery vehicle of one of aboveembodiments, wherein the peptide in the polymer is resistant toproteolytic degradation.21a. The composition or the drug delivery vehicle of one of aboveembodiments, forming a spherical micellar assembly.22a. The composition or the drug delivery vehicle of one of aboveembodiments, comprising a side chain protecting group.23a. The composition or the drug delivery vehicle of one of aboveembodiments, comprising a detectable moiety.24a. The composition or the drug delivery vehicle of one of aboveembodiments, wherein the detectable moiety is a poly-histidine(poly(His)), chitin binding protein, maltose binding protein,glutathione-S-transferase (GST), FLAG-tag, AviTag, Calmodulin-tag,polyglutamate tag, E-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softtag 1,Softtag 3, Strep-tag, TC tag, V5 tag, VSV tag, Xpress tag, Isopeptag,SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tag, Halo-tag,thioredoxin-tag, or Fc-tag, electron-dense reagent, enzyme, biotin,digoxigenin, paramagnetic molecule, paramagnetic nanoparticle, contrastagent, magnetic resonance contrast agent, X-ray contrast agent,Gadolinium, radioisotope, radionuclide, fluorodeoxyglucose, gamma rayemitting radionuclide, positron-emitting radionuclide, biocolloid,microbubble, iodinated contrast agent, barium sulfate, thorium dioxide,gold, gold nanoparticle, gold nanoparticle aggregate, fluorophore,two-photon fluorophore, hapten, protein, or fluorescent moiety.25a. A method of preparing a cell penetrating high density brush peptidepolymer, wherein the peptide polymer comprises peptides with an arginineor a lysine residue appended to the N- or at the C-terminus, and ispendant to a polymerized norbornene moieties, comprising combining aplurality of the peptide monomer with a ruthenium catalyst into amixture for ring opening metathesis polymerization (ROMP).26a. The method of any previous embodiment, wherein the polymer is ahomopolymer or a block copolymer.27a. The method of any previous embodiment, wherein the block copolymeris prepared by first polymerizing the peptide monomer to completionprior to adding and polymerizing the non-peptidic monomer.28a. A method of delivering a peptide, pharmaceutically active agent, oran oligonucleotide into a cell by the drug delivery vehicle of anyprevious embodiment.

As used herein, the term “Drug delivery vehicle or system” is used inaccordance with its common meaning in the chemical sciences and refersto high density brush peptide polymers disclosed herein, that used forthe targeted delivery and/or controlled release of therapeutic agentsinto an interior of a cell. In embodiments, the drug delivery vehiclesof the present disclosure including an active therapeutic agent areadministered as pharmaceutical compositions.

1. A polymer having the formula: R¹-[M(O)]_(n)—R² wherein, n is aninteger from 2 to 1000; M is a polymerized monomer; O is independently atherapeutic polypeptide (a therapeutic polypeptide moiety) covalentlyattached to M through a covalent linker; and R¹ and R² are independentlyterminal polymer moieties.2. A block copolymer having the formula: R¹-[M(O)]_(n)-[M(P)]_(m)—R² orR¹-[M(P)]_(m)-[M(O)]_(n)—R² wherein, n is an integer from 2 to 1000; mis an integer from 2 to 1000; M is a polymerized monomer; O isindependently a polypeptide (a polypeptide moiety) covalently attachedto M through a covalent linker; P is independently a non-polypeptidemoiety; and R¹ and R² are independently terminal polymer moieties.3. A blend copolymer having the formula:R¹-([M(O)]_(n)-[M(P)]_(m))_(z)—R² or R¹-([M(P)]_(m)-[M(O)]_(n))_(z)—R²wherein, n is an integer from 2 to 1000; m is an integer from 2 to 1000;M is a polymerized monomer; O is independently a polypeptide (apolypeptide moiety) covalently attached to M through a covalent linker;P is independently a non-polypeptide moiety; z is an integer from 2 to100; and R¹ and R² are independently terminal polymer moieties.4. The copolymer of one of embodiments 2 to 3, wherein saidnon-polypeptide moiety is independently a substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl and wherein said non-polypeptide moiety does not comprise anucleotide.5. The copolymer of one of embodiments 2 to 3, wherein said polypeptideis independently a therapeutic polypeptide.6. The copolymer of one of embodiments 2 to 3, wherein saidnon-polypeptide moiety is independently a hydrophobic moiety.7. The polymer of one of embodiments 1 to 6, wherein the polymerizablemonomer is N-substituted-5-norbornene-2,3-dicarboximide, wherein thesubstitution independently comprises the therapeutic polypeptide.8. The polymer of one of embodiments 1 to 6, wherein M(O) is

L¹ is is independently a bond, —O—, —NH—, —COO—, —S—, —SO₂—, —SO₃—,—SO₄—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —NHC(O)O—, NHC(O)NH—, C(O),substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene; and R⁴ isindependently a therapeutic polypeptide.9. The polymer of one of embodiments 1 to 8, wherein the polypeptidecomprises from 2 to 6 arginine residues.10. The polymer of one of embodiments 1 to 8, wherein the polypeptidecomprises 2 arginine residues.11. The polymer of one of embodiments 9 to 10, wherein the arginineresidues are the carboxyl terminal residues of the polypeptide.12. The polymer of one of embodiments 1 to 11, wherein the polypeptidedoes not comprise a carboxy terminal lysine residue.13. The polymer of one of embodiments 1 to 12, wherein the polypeptideis resistant to proteolysis relative to the unpolymerized polypeptide.14. The polymer of one of embodiments 1 to 13, wherein R¹ independentlycomprises a substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl.15. The polymer of one of embodiments 1 to 14, wherein R² independentlycomprises a substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl.16. The polymer of one of embodiments 1 to 15, wherein the polymercomprises a detectable moiety.17. A micelle comprising the copolymer of one of embodiments 2 to 16.18. The micelle of embodiment 17, having a longest dimension (e.g.,diameter) of between about 1 and about 1000 nm.19. The micelle of embodiment 17, having a longest dimension (e.g.,diameter) of between about 5 and about 100 nm.20. The micelle of embodiment 17, having a longest dimension (e.g.,diameter) of between about 10 and about 50 nm.21. A nanoparticle comprising the copolymer of one of embodiments 2 to16.22. The nanoparticle of embodiment 21, wherein said nanoparticle is aspherical nanoparticle.23. The nanoparticle of one of embodiments 21 to 22, having a longestdimension (e.g., diameter) of between about 1 and about 1000 nm.24. The nanoparticle of one of embodiments 21 to 22, having a longestdimension (e.g., diameter) of between about 5 and about 100 nm.25. The nanoparticle of one of embodiments 21 to 22, having a longestdimension (e.g., diameter) of between about 10 and about 50 nm.26. A method of administering a polypeptide to the interior of a cellcomprising contacting said cell with the polymer of one of embodiments 1to 16; the micelle of one of embodiments 17 to 20; or the nanoparticleof one of embodiments 21 to 25.27. The method of embodiment 26, wherein said cell is in a subject.28. The method of embodiment 27, wherein said polymer, micelle, ornanoparticle is administered systemically to said subject.29. A method of treating a disease in a subject, comprisingadministering to said subject a polymer of one of embodiments 1 to 16;the micelle of one of embodiments 17 to 20; or the nanoparticle of oneof embodiments 21 to 25.

EXAMPLES Example 1: Polymerization of Cell Penetrating Peptides

Materials

The following materials were used to generate the data described herein.Amino acids used in SPPS were purchased from Aapptec and NovaBiochem.All other materials were obtained from Sigma-Aldrich and used withoutfurther purification unless otherwise noted. Initiator 1((H₂IMES)(pyr)₂(Cl)₂Ru═CHPh) was prepared. Analytical scale RP-HPLC wasperformed with a Jupiter Proteo90A Phenomenex column (150×4.60 mm) usinga Hitachi-Elite LaChrom L2130 pump with a UV-vis detector (Hitachi-EliteLaChrome L-2420) monitoring at 214 nm. Peptides were purified with aJupiter Proteo90A Phenomenex column (2050×25.0 mm) on a Waters DeltaPrep300 System. For all RP-HPLC assays, gradient solvent systems were usedin which Buffer A was 0.1% TFA in water and Buffer B was 0.1% TFA inacetonitrile. Polymer dispersities and molecular weights were determinedby size-exclusion chromatography (Phenomenex Phenogel 5 u 10, 1-75 K,300×7.80 mm in series with a Phenomenex Phenogel 5 u 10, 10-1000 K,300×7.80 mm with 0.05 M LiBr in DMF as the running buffer at a flow rateof 0.75 mL/min) using a Shimadzu pump equipped with a multiangle lightscattering detector (DAWN-HELIO, Wyatt Technology) and a refractiveindex detector (HITACHI L2490 or a Wyatt Optilab T-rEX detector)normalized to a 30 K MW polystyrene standard. For SEC-MALS chromatogramsin which a multimodal distribution is observed by light scattering butnot in the RI chromatogram, only the peak width that has an associatedRI component was analyzed. DLS measurements were performed on a DynaProNanoStar (Wyatt Tech). TEM images were obtained by depositing samples oncarbon-formavar-coated copper grids (Ted Paella, Inc.), which were thenstained with 1% w/w uranyl acetate and then imaged on a Techanai G2Sphera operating at an accelerating voltage of 200 kV. Allconcentrations of fluorescent materials were obtained by measuring UVabsorbance of the fluorophore on a ThermoScientific Nanodrop 2000c andthe data was fit to the standard curves. Fluorescent data was recordedon a fluorescence plate reader, PerkinElmer HTS 7000 Plus Bio AssayReader (excitation: 340 nm; emission: 465 nm), or on a Photon TechnologyInternational fluorescence reader. 1H (400 MHz) and 13C (100 MHz) NMRspectra were recorded on a Varian Mercury Plus spectrometer. Chemicalshifts are reported in ppm relative to the DMF-d7 or CDCl3 residualpeaks.

SEC-MALS chromatograms was obtained for GSGSG polymers at a peptide m of˜60 in DMF with 0.05 M LiBr. In some traces, a multimodal distributionis seen by LS, but only a monomodal distribution is observed at t>15 min the RI component. In these scenarios, only the peak width with anassociated RI component is used to calculate values. Note that theGSGSGK monomer with side chain protecting groups formed a gel uponpolymerization, which made it difficult to run these samples on the SECcolumn. A saturated solution of LiBr was added to break up the gel toachieve the traces shown. The deprotected polymer did not aggregate inDPBS and showed no signal by dynamic light scattering, suggesting thatno larger aggregates were present. SEC-MALS chromatograms were obtainedfor R control polymer at a peptide m of ˜60 in DMF with 0.05 M LiBr.SEC-MALS chromatograms were obtained for RGSGSG polymer at a peptide mof ˜60 in DMF with 0.05 M LiBr. SEC-MALS chromatograms were obtained forRRGSGSG polymer at a peptide m of ˜60 in DMF with 0.05 M LiBr. SEC-MALSchromatograms were obtained for RGSGSG polymer at a peptide m of ˜60 inDMF with 0.05 M LiBr. SEC-MALS chromatograms were obtained for forGSGSGRR polymer at a peptide m of ˜60 in DMF with 0.05 M LiBr. SEC-MALSchromatograms were obtained for GSGSGK polymer at a peptide m of ˜60 inDMF with 0.05 M LiBr.

RP-HPLC chromatatograms were obtained for:Norgly-E(EDANS)RPAHLRDSGK(dabcyl)GSGSG-NH₂ (SEQ ID NO:6) gradient at0-67% Buffer B; NorGly-K(dabcyl)RPAHLRDSGE(EDANS)GSGSG-NH₂ (SEQ IDNO:8), gradient at 0-67% Buffer B;NorGly-GSGSGE(EDANS)RPAHLRDSGK(dabcyl)NH₂ (SEQ ID NO: 10), gradient at10-67% Buffer B; the C-terminal fragment H-RPAHLRDSGK(dabcyl)GSGSG-NH₂,gradient at 0-67% Buffer B; and the N-terminal fragmentNorGly-E(EDNAS)RPAH-H, gradient at 10-30% Buffer B. Other enzymes usedherein cleave cleave the substrate at different locations. The C- andN-terminal chromatograms were used to generate standard curves for allkinetic assays.

A spectrum monitoring the polymerization by ¹HNMR ofNorGly-E(EDANS)RPAHLRDSGK(dabcyl)GSGSG-NH₂ was obtained. The absence ofthe olefenic proton from the monomer (seen at δ 6.32 ppm in the spectrumof the monomer) after 3 hours of polymerization and the correspondingappearance of two broad peaks from approximately δ 5.4-6 ppm signify thecis- and trans-olefin protons of the norbornyl polymer backbone. Aspectrum monitoring the polymerization by ¹HNMR ofNorGly-K(dabcyl)RPAHLRDSGE(EDANS)GSGSG-NH₂ was obtained. The absence ofthe olefenic proton from the monomer (seen at δ 6.32 ppm in the spectrumof the monomer) after 3 hours of polymerization and the correspondingappearance of two broad peaks from approximately δ 5.4-6 ppm signify thecis- and trans-olefin protons of the norbornyl polymer backbone. Aspectrum monitoring the polymerization by ¹HNMR ofNorGly-GSGSGE(EDANS)RPAHLRDSGK(dabcyl)NH₂ was obtained. The absence ofthe olefenic proton from the monomer (seen at δ 6.32 ppm in the spectrumof the monomer) after 3 hours of polymerization and the correspondingappearance of two broad peaks from approximately δ 5.4-6 ppm signify thecis- and trans-olefin protons of the norbornyl polymer backbone.

Validation of the proteolytic resistance and bioactivity of polymerizedCPPs (canonical cell-penetrating peptides) required preparation ofwell-defined brush polymers with low dispersity via a livingpolymerization method. High-density brush polymers of knowncell-penetrating peptides, Tat and Arg8, together with appropriatecontrol polymers, were prepared via living ROMP (ring opening metathesispolymerization) by an initiator, ((H₂IMES)-(pyr)₂(Cl)₂Ru═CHPh) (FIG.1A). ROMP by this initiator was selected for preparation of thesematerials for a variety of reasons. First, the initiator exhibits fastinitiation and slower propagation kinetics, which typically affordpolymers with exceptionally narrow molecular weight distributions.Second, it is highly functional group tolerant, enabling theincorporation of a wide range of chemical functionality viapolymerization of groups pendant to a norbornene moiety, includingfluorophores, drugs, sugars, oligonucleotides, and peptides. Very fewpolymerization techniques have been shown to incorporate peptidesdirectly by graft-through polymerization from a peptide-containingmonomer. Reports on graft-through polymerization of peptides byreversible addition-fragmentation chain transfer (RAFT) or free-radicalpolymerizations describe only blend polymers with less than 50%incorporation of peptides. Additionally, the polymers produced by thesemethods generally have broader molecular weight distributions than thosetypically afforded by ROMP. Furthermore, a high degree of functionalityand complexity can be readily generated on a single polymer via ROMP bypreparing multiblock copolymers of appropriately functionalizednorbornene monomers or via the use of chain transfer agents to end-labela polymer through a single cross metathesis event upon completeconsumption of monomers.

Guanidinium Monomer Synthesis Methods

A 10 mL round-bottom flask equipped with a stir bar was charged with anamine-terminated norbornene(2-(2-aminoethyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoi-soindole-1,3-(2H)-dione)(70 mg, 0.24 mmol, 1 equiv), which was prepared and dissolved in 4.5 mLof dry DMF under N₂ (g). To this was added N,N-bis(boc)-1-guanylpyrazole(105 mg, 0.34 mmol, 1 equiv) and diisopropylethylamine (120 μL, 0.68mmol, 2 equiv). The reaction mixture was stirred at room temperature for12 h. The solution was concentrated to dryness and re-suspended in 25 mLof CH₂Cl₂, then washed with water (×3) and then brine. The CH₂Cl₂ layerwas collected, dried over Na₂SO₄ (s) and concentrated to dryness. Thematerial was then purified by flash column chromatography on silica gel(33% EtOAC in hexanes) to yield a white powder in 92% yield (140 mg,0.31 mmol) R_(f)0.37 (33% EtOAc in hexanes): ¹H NMR (400 MHz, CDCl₃, 298K) δ11.43 ppm (1H, b), 8.45 (1H, b), 6.27 (2H, t, J=1.5 Hz), 3.71 (2H,dd, J=7.0, 4.4), 3.64 (2H, m), 3.25 (2H, d, J=1.5 Hz), 2.7 (2H, m), 1.51(1H, d, J=1.1 Hz), 1.48 (9H, s), 1.47 (9H, s), 1.25 (1H, d, J=1.8 Hz);¹³C NMR (100 MHz, CDCl₃, 298 K) δ 178.1, 157.0, 156.6, 153.0, 137.9,137.7, 83.3, 79.5, 48.0, 45.0, 43.1, 40.0, 38.0, 28.3, 28.1;High-resolution MS analysis (ESI-TOFMS) m/z calculated 449.2395, found449.2394.

Peptide Synthesis Methods

Peptides were synthesized using standard Solid Phase Peptide Synthesis(SPPS) procedures on an AAPPTec Focus XC automated synthesizer. The Arg8peptide was synthesized with the Pbf protecting group left on the sidechains by the use of highly acid-sensitive Sieber Amide resin. Mostpeptide monomers were synthesized to contain amino acid side chainprotecting groups by the use of the highly acid-sensitive Sieber Amideresin, which allows for cleavage of the peptide from the resin withoutremoval of the protecting groups. All other peptides were preparedprotecting group-free via the use of Rink Amide MBHA resin.

A typical SPPS procedure involved FMOC deprotection with 20%methyl-piperidine in DMF (one 5 min deprotection followed by one 15 mindeprotection), and 45 min amide couplings using 3.75 equiv of theFMOC-protected, and side chain-protected amino acid, 4 equiv of HBTU and8 equiv of DIPEA. Peptide couplings that were incomplete by Kaiser Testwere drained and then subjected to fresh reagents. Monomers wereprepared by amide coupling to N-(hexanoicacid)-cis-5-norbornene-exo-dicarboximide or toN-(glycine)-cis-5-norbornene-exo-dicarboximide for the fluorogenicsubstrates at the N-terminus of the peptide.

The “R control” was prepared by conjugating Arg toN-(glycine)-cis-5-norbornene-exo-dicarboximide. The N-glycine derivativewas used to produce a shorter linker between the Arg residue and thenorbornene unit to provide as little flexibility as possible. Data fromthis control is, thus, taken to reflect the maximum theoretical uptakethat should be achieved by a peptide containing a single Arg.

Fluorescein-labeled peptides were assembled by addition ofBoc-Lys(FMOC)-OH to the N-terminus of the peptide, followed by removalof the FMOC protecting group and amide coupling to 5/6-carboxyfluorescein. Following completion of the synthesis, peptides werecleaved from the resin. Following completion of the synthesis, peptideswere cleaved from the resin. All side-chain protected peptide monomerswere cleaved from the Sieber amide resin by five consecutive rinses with2% TFA in DCM for two minutes each. All other peptides were cleaved fromRink MBHA resin and deprotected by treatment with TFA/H2O/TIPS in a9.5:2.5:2.5 ratio for 2 hrs. The peptides were then precipitated in coldether and purified by RP-HPLC. The purity of each peptide was verifiedby analytical RP-HPLC, where a single peak in the chromatogram of anewly purified peptide was taken as an indication of a pure material.The identity of each peptide was confirmed by ESI MS (Table 1 and Table2). The side-chain protected Arg8 peptide was cleaved from the Sieberamide resin by five 2 min rinses with 2% TFA in DCM. A chromatogramshowing the purity of the NorAhaR(Pbf)GS(OtBu)GS(OtBu)G-NH₂ peptide at30-50% Buffer B was obtained. The identity of the peak was confirmedusing ESI MS shown in Table 1. A chromatogram showing the purity of theNorAhaR(Pbf)R(Pbf)GS(OtBu)GS(OtBu)G-NH₂ peptide at 40-60% Buffer B wasobtained. The identity of the peak was confirmed using ESI MS shown inTable 1. A chromatogram showing the purity of theNorAhaGS(OtBu)GS(OtBu)GR(Pbf)-NH₂ peptide at 30-50% Buffer B wasobtained. The identity of the peak was confirmed using ESI MS shown inTable 1. A chromatogram showing the purity of theNorAhaGS(OtBu)GS(OtBu)GR(Pbf)R(Pbf)-NH₂ peptide at 40-60% Buffer B wasobtained. The identity of the peak was confirmed using ESI MS shown inTable 1. A chromatogram showing the purity of theNorAhaGS(OtBu)GS(OtBu)GK(Boc)-NH₂ peptide at 30-90% Buffer B wasobtained. The identity of the peak was confirmed using ESI MS shown inTable 1. A chromatogram showing the purity of the NorAhaGSGSGKK-NH₂peptide at 10-50% Buffer B was obtained. The identity of the peak wasconfirmed using ESI MS shown in Table 1. A chromatogram showing thepurity of the NorGlyR-NH₂ peptide at 30-75% Buffer B was obtained. Theidentity of the peak was confirmed using ESI MS shown in Table 1. AnRP-HPLC chromatogram confirming the purity of) NorAhaKLAKLAKKLAKLAK-NH₂(full length KLA) at 0-50% Buffer B was obtained. NorAha=N-(hexanoicacid)-cis-5-norbornene-exo-dicarboximide; The identity of each peak wasconfirmed by ESI MS as shown in Table 1. An RP-HPLC chromatogramconfirming the purity NorAhaKLAKLAK-NH₂ (fragment KLA) at 10-40% BufferB was obtained. NorGly ═N-(glycine)-cis-5-norbornene-exo-dicarboximide.Note that some peptides were synthesized with protecting groups on theamino acid side chains. The identity of each peak was confirmed by ESIMS as shown in Table 1. An RP-HPLC chromatogram confirming the purity ofK(Flu)RGSGSG was obtained. The peptide was purified using a gradient of0-67% Buffer B. The identity of each peak was confirmed by ESI MS asshown in Table 2. Flu represents 5/6-carboxyfluorescein, which isconjugated to the ε amino group of the N-terminal Lys on each peptide.An RP-HPLC chromatogram confirming the purity of K(Flu)RRGSGSG wasobtained. The peptide was purified using a gradient of 0-67% Buffer B.The identity of each peak was confirmed by ESI MS as shown in Table 2.An RP-HPLC chromatogram confirming the purity of K(Flu)GSGSGR wasobtained. The peptide was purified using a gradient of 0-67% Buffer B.The identity of each peak was confirmed by ESI MS as shown in Table 2.An RP-HPLC chromatogram confirming the purity of K(Flu)GSGSGRR wasobtained. The peptide was purified using a gradient of 0-67% Buffer B.The identity of each peak was confirmed by ESI MS as shown in Table 2.An RP-HPLC chromatogram confirming the purity of K(Flu)GSGSGK wasobtained. The peptide was purified using a gradient of 0-67% Buffer B.The identity of each peak was confirmed by ESI MS as shown in Table 2.An RP-HPLC chromatogram confirming the purity of K(Flu)GSGSGKK wasobtained. The peptide was purified using a gradient of 0-67% Buffer B.The identity of each peak was confirmed by ESI MS as shown in Table 2.An RP-HPLC chromatogram confirming the purity of K(Flu)KLAKLAKKLAKLAKwas obtained. The peptide was purified using a gradient of 0-67% BufferB. The identity of each peak was confirmed by ESI MS as shown in Table2.

A standard curve, correlating peak area on RP-HPLC chromatograms withconcentration on an 18 μL injection for the determination of theconcentration of intact KLA peptide remaining after proteolytic cleavagewas obtained. Percent proteolytic cleavage is given in FIG. 21B. Theconcentration is with respect to the peptide content. A standard curve,correlating peak area on RP-HPLC chromatograms with concentration on an18 μL injection for the determination of the concentration of intact KLApolymer (DP˜10) remaining after proteolytic cleavage was obtained.Percent proteolytic cleavage is given in FIG. 21B. The concentration iswith respect to the peptide content. A standard curve, correlating peakarea on RP-HPLC chromatograms with concentration on an 18 μL injectionfor the determination of the concentration of intact GSGSGRR peptideremaining after proteolytic cleavage. Percent proteolytic cleavage isgiven in FIG. 21B. The concentration is with respect to the peptidecontent. A standard curve, correlating peak area on RP-HPLCchromatograms with concentration on an 18 μL injection for thedetermination of the concentration of intact GSGSGRR polymer (DP˜60)remaining after proteolytic cleavage was obtained. Percent proteolyticcleavage is given in FIG. 21B. The concentration is with respect to thepeptide content. A standard curve, correlating peak area on RP-HPLCchromatograms with concentration on an 18 μL injection for thedetermination of the concentration of intact GSGSGKK peptide remainingafter proteolytic cleavage was obtained. Percent proteolytic cleavage isgiven in FIG. 21B. The concentration is with respect to the peptidecontent. A standard curve, correlating peak area on RP-HPLCchromatograms with concentration on an 18 μL injection for thedetermination of the concentration of intact GSGSGKK polymer (DP˜60)remaining after proteolytic cleavage was obtained. Percent proteolyticcleavage is given in FIG. 21B. The concentration is with respect to thepeptide content.

A trace of a RP-HPLC assay for the proteolytic cleavage of theK(Flu)GSGSGRR peptide with no pronase was obtained. Similarly, a traceof a RP-HPLC assay for the proteolytic cleavage of the GSGSGR polymer(m˜60) with no pronase was obtained. Similarly, a trace of a RP-HPLCassay for the proteolytic cleavage of the K(Flu)GSGSGRR peptide withtrypsin was obtained. The peptide (50 μM) was incubated with trypsin (1μM) for 3 hours in DPBS. The identity of the major peptide fragmentswere determined by ESI MS. Similarly, a trace of a RP-HPLC assay for theproteolytic cleavage of the GSGSGRR polymer (m˜60) with trypsin wasobtained. The peptide (50 μM) was incubated with trypsin (1 μM) for 3hours in DPBS. The identity of the major peptide fragments weredetermined by ESI MS. Similarly, a trace of a RP-HPLC assay for theproteolytic cleavage of the K(Flu)GSGSGRR peptide with a pronase(cocktail) was obtained. The peptide (50 μM) was incubated with thepronase cocktail (1 μM) for 3 hours in DPBS. The identity of the majorpeptide fragments were determined by ESI MS. Similarly, a trace of aRP-HPLC assay for the proteolytic cleavage of the GSGSGRR polymer (m˜60)with a pronase (cocktail) was obtained. The peptide (50 μM) wasincubated with the pronase cocktail (1 μM) for 3 hours in DPBS. Theidentity of the major peptide fragments were determined by ESI MS.Similarly, a trace of a RP-HPLC assay for the proteolytic cleavage ofthe K(Flu)GSGSGKK peptide with no pronase was obtained. Similarly, atrace of a RP-HPLC assay for the proteolytic cleavage of the GSGSGKKpolymer (m˜60) with no pronase was obtained. Similarly, a trace of aRP-HPLC assay for the proteolytic cleavage of the K(Flu) GSGSGKK peptidewith trypsin was obtained. The peptide (50 μM) was incubated withtrypsin (1 μM) for 3 hours in DPBS. The identity of the major peptidefragments were determined by ESI MS. Similarly, a trace of a RP-HPLCassay for the proteolytic cleavage of the GSGSGKK polymer (m˜60) withtrypsin was obtained. The peptide (50 μM) was incubated with trypsin (1μM) for 3 hours in DPBS. The identity of the major peptide fragmentswere determined by ESI MS. Similarly, a trace of a RP-HPLC assay for theproteolytic cleavage of the K(Flu) GSGSGKK peptide with a pronase(cocktail) was obtained. The peptide (50 μM) was incubated with thepronase cocktail (1 μM) for 3 hours in DPBS. The identity of the majorpeptide fragments were determined by ESI MS. Similarly, a trace of aRP-HPLC assay for the proteolytic cleavage of the GSGSGKK polymer (m˜60)with a pronase (cocktail) was obtained. The peptide (50 μM) wasincubated with the pronase cocktail (1 μM) for 3 hours in DPBS. Theidentity of the major peptide fragments were determined by ESI MS.Similarly, a trace of a RP-HPLC assay for the proteolytic cleavage ofthe K(Flu)KLAKLAKKLAKLAK peptide with no pronase was obtained.Similarly, a trace of a RP-HPLC assay for the proteolytic cleavage ofthe KLAKLAKKLAKLAK polymer (m˜10) with no pronase. Similarly, a trace ofa RP-HPLC assay for the proteolytic cleavage of the K(Flu)KLAKLAKKLAKLAK peptide with trypsin was obtained. The peptide (50 μM)was incubated with trypsin (1 μM) for 3 hours in DPBS. The identity ofthe major peptide fragments were determined by ESI MS. Similarly, atrace of a RP-HPLC assay for the proteolytic cleavage of theKLAKLAKKLAKLAK (m˜10) polymer with trypsin was obtained. The peptide (50μM) was incubated with trypsin (1 μM) for 3 hours in DPBS. The identityof the major peptide fragments were determined by ESI MS. Similarly, atrace of a RP-HPLC assay for the proteolytic cleavage of the K(Flu)KLAKLAKKLAKLAK peptide with a pronase (cocktail) was obtained. Thepeptide (50 μM) was incubated with the pronase cocktail (1 μM) for 3hours in DPBS. The identity of the major peptide fragments weredetermined by ESI MS. Similarly, a trace of a RP-HPLC assay for theproteolytic cleavage of the KLAKLAKKLAKLAK (m˜10) polymer with a pronase(cocktail) was obtained. The peptide (50 μM) was incubated with thepronase cocktail (1 μM) for 3 hours in DPBS. The identity of the majorpeptide fragments were determined by ESI MS.

TABLE 1 Molecular masses obtained for each peptide monomer. Cal-Obtained culated Mass Peptide Monomer Mass (ESI)NorAhaR(Pbf)GS(OtBu)GS(OtBu) 1142 1143 G-NH₂ (SEQ ID NO: 44)NorAhaR(Pbf)R(Pbf)GS(OtBu)GS 1550 1552 (OtBu)G-NH₂ (SEQ ID NO: 45)NorAhaGS(OtBu)GS(OtBu)GR 1142 1143 (Pbf)-NH₂ (SEQ ID NO: 46)NorAhaGS(OtBu)GS(OtBu)GR(Pbf) 1550 1552 R(Pbf)-NH₂ (SEQ ID NO: 47)NorAhaGS(OtBu)GS(OtBu)GK  964  963 (Boc)-NH₂ (SEQ ID NO: 48)NorAhaGSGSGKK-NH₂  879  878 (SEQ ID NO: 49) NorAhaKLAKLAKKLAKLAK-NH₂1781 1782 (full length)(SEQ ID NO: 50) NorAhaKLAKLAK-NH₂ 1029 1027(fragment)(SEQ ID NO: 51) NorG1yR(Pbf)-NH₂  628  627

-   -   Note that several sequences were synthesized with protecting        groups on the amino acid side chains. Masses were obtained by        ESI MS and confirm the identity of each monomer.

TABLE 2 Molecular masses obtained for eachfluorescently-labeled peptide control. Cal- Obtained culated MassPeptide Control Mass (ESI) K(Flu)RGSGSG-NH₂ 1005 1004 (SEQ ID NO: 52)K(Flu)RRGSGSG-NH₂ 1161 1160 (SEQ ID NO: 53) K(Flu)GSGSGR-NH₂ 1005 1004(SEQ ID NO: 54) K(Flu)GSGSGRR-NH₂ 1161 1160 (SEQ ID NO: 55)K(Flu)GSGSGK-NH₂  977  978 (SEQ ID NO: 56) K(Flu)GSGSGKK-NH₂ 1106 1105(SEQ ID NO: 57) K(Flu)KLAKLAKKLAKLAK-NH₂ 2011 2010 (SEQ ID NO: 58)

-   -   All sequences are prepared with an N-terminal Lys that is        conjugated to 5/6-carboxyfluorescein via the ε-amino group.        Masses were obtained by ESI MS and confirm the identity of each        monomer.

TABLE 3 Calculated and obtained molecular weightvalues of peptides prepared for cell penetrationstudies. Sequence legend (in order of appearance):SEQ ID NO: 59-64, respectively. Mass Cal- Mass Peptide Sequence culatedObtained Flu-Tat K(5/6-Flu)YGRKKRRQR 2045.4  2044.8 Peptide RR-NH₂Flu-Arg8 K(5/6-Flu)RRRRRRRR- 1753.02 1752.1 Peptide NH₂ Flu-GSGSGK(5/6-Flu)GSGSG-NH₂  848.82  849.1 Peptide NorAha-TATNorAha-YGRKKRRQRRR- 1818.9  1818.5 Monomer NH₂ NorAha-Arg8NorAha-R(Pbf)R(Pbf) 3541.6  3542.4 Monomer R(Pbf)R(Pbf)R(Pbf)R(Pbf)R(Pbf)R(Pbf)- NH₂ NorAha-GSGSG NorAha-GSGSG-NH₂  622.3   623.3Monomer

-   -   Tat and Arg8 masses were obtained via MALDI-TOF MS and GSGSG        masses were taken on ESI (+) MS.

TABLE 4 Characterization data for each homopolymer containing thethrombin peptide substrate as determined by batch mode SLS (static lightscattering) in a cuvette. Polymer M_(n) DP Thrombin Homopolymer (10 mer)16,800 11 (10) Thrombin Homopolymer (20 mer) 33,300 23 (20) ThrombinHomopolymer (30 mer) 43,900 30 (30)

TABLE 5 Amino acid sequences and molecular weights calculated and obtained for fluorogenic monomersand authentic products. Molecular weights wereobtained by ESI (+) MS. Sequence legend (order ofappearance): SEQ ID NOs: 6, 8, 10, 65 and 66, respectively. Mass Cal-Mass Peptide Sequence culated Obtained FluorogenicNorGly-E(EDANS)RPAHLRD 2311.03 2312.9 monomer SGK(dabcyl)GSGSG-NH₂Reversed NorGly-K(dabcyl)RPAHLR 2311.03 2312.8 FluorogenicDSGE(EDANS)GSGSG-NH₂ Monomer Fluorogenic NorGly-GSGSGE(EDANS)RP 2311.032313.7 Monomer AHLRDSGK(dabcyl)-NH₂ with linker N-terminalNorGly-E(EDANS)RPAH-H 1059.42 1060.6 Fragment C-terminalH-LRDSGK(dabcyl)GSGSG- 1269.5  1270.6 Fragment NH₂

TABLE 6 Theoretical and experimentally determined M_(n) values forfluorogenic homopolymers. M_(n) is obtained by batch mode SLS with acuvette. Theoretical Polymer M_(n) M_(n) DP Fluorogenic Homopolymer46,000 43,000 19 Reversed Fluorogenic Homopolymer 46,000 43,000 19Fluorogenic Homopolymer with Linker 46,000 45,000 20

Polymerizations Methods

Polymerizations were carried out in a glovebox under N₂ (g). A typicalprotocol used to generate a polymer with DP=10 involved mixing themonomer (0.0125 mmol, 10 equiv, 25 mM) with the catalyst (0.00125 mmol,1 equiv, 2.5 mM) in dry DMF (0.5 mL). Homopolymerizations were performedin DMF-d7 and followed by 1H NMR to confirm complete consumption of themonomer and to determine the time period required to reach completion.Polymers for cell penetration studies were end-labeled with a copy offluorescein using a chain transfer agent (1.5 equiv) for 2 h, followedby termination with ethyl vinyl ether (10 equiv) for 1 h at roomtemperature. Block copolymers were prepared by polymerizing the firstmonomer (either phenyl or PEG) to completion and then adding the secondmonomer (a peptide or the guanidinium group), followed by end labelingwith the fluorescein chain transfer agent and finally termination withethyl vinyl ether. Fluorescein-labeled polymers were treated with NH₄OH(aq) for 20 min to remove the pivolate protecting group. The resultingpolymers were directly characterized by SEC-MALS.

The sidechain protected Arg8 polymer was precipitated with cold etherand collected by centrifugation. The resulting powder was dissolved in 2mL of a mixture of TFA/H2O/TIPS (95:2.5:2.5) and stirred for 4 h at roomtemperature. The product was precipitated with cold ether, collected bycentrifugation and dried. In preparation for in vitro studies, allpolymers were washed (×3) with cold ether (to remove the Ru catalyst)and then dissolved in PBS and dialyzed in an effort to remove anyresidual monomer or catalyst. The Tat and GSGSG (SEQ ID NO: 1) particleswere generated by dissolving the amphiphilic polymers in DMF, and thendiluting with an equivalent volume of PBS over 1 hand finally dialyzingthis solution into PBS over 48 h with 3 buffer changes using dialysiscups of MWCO 3500 (Thermo Scientific, cat. #69552).

Peptides used to generate ROMP monomers were prepared by solid phasepeptide synthesis (SPPS) using standard fluorenylmethyloxycarbonyl(FMOC) chemistry. Peptide monomers were prepared by coupling acarboxylic acid-modified norbornene to the N-terminus of the desiredpeptide sequence on resin. Peptide monomers with side chain protectinggroups and a five-carbon linker between the peptide and the norborneneunit generally polymerize at faster rates than those with shorterlinkers or those that possess no protecting groups. Therefore, allpeptide monomers were prepared with this linker and used an Arg8 monomerwith side chains protected. However, efforts to prepare and polymerizethe protected Tat peptide were thwarted by poor solubility of theprotected material in solvents compatible with the ROMP initiator.Therefore, the Tat peptide monomer was prepared without protectinggroups (FIG. 12 for chemical structures of monomers). All peptides thatwere incorporated into polymers were also separately prepared (withoutnorbornyl-groups) as fluorescein-labeled peptides via conjugation of5/6-carboxyfluorescein to the ε-amino group of an N-terminal lysine, foruse as controls to evaluate the cellular uptake efficiency of thepeptides alone versus polymerized materials. (Table 3)

HPLC chromatogram of purified peptide monomer NorAha-RRRRRRRR-NH₂(NorAha-Arg8, SEQ ID NO:67) at 80-100% B gradient was obtained,demonstrating a single peak at about 26 min. HPLC chromatogram ofpurified peptide monomer NorAhaYGRKKRRQRRR-NH₂ (NorAha-Tat, SEQ IDNO:68) at 10-40% B gradient was obtained, demonstrating a major peak atabout 17 min. HPLC chromatogram of purified peptide monomerNorAha-GSGSG-NH₂ (NorAha-GSGSG, SEQ ID NO:69) at 0-67% B gradient wasobtained, demonstrating a major peak at about 18 min. HPLC chromatogramof purified peptide monomer K(Fluorescein)-RRRRRRRR-NH₂ (Flu-Arg8, SEQID NO:70) at 0-67% B gradient was obtain, demonstrating a major peak atabout 20.5 min. HPLC chromatogram of purified peptide monomerK(Fluorescein)-YGRKKRRQRR-NH2 (Flu-Tat, SEQ ID NO:71) at 0-67% Bgradient was obtained, demonstrating a major peak at about 17 min. HPLCchromatogram of purified peptide K(Fluorescin)-GSGSG-NH₂ (Flu-GSGSG, SEQID NO:72) at 0-67% B gradient) was obtained, demonstrating a major peakat about 18.5 min. ¹H NMR and ¹³C NMR spectra of a guanidinium monomerwere obtained.

Characterization of Polymerized Peptides

Each peptide-based monomer was polymerized, and the resulting polymerswere end-labeled with fluorescein to enable tracking of the uptake ofthe material and to serve as a model cargo (FIG. 1A). In addition topolymers containing the canonical Tat and Arg8 CPPs, several controlpolymers were prepared, including a polymer of an uncharged, nonpeptideunit of oligoethylene glycol (OEG) and another consisting of a peptideside chain that did not contain any charged residues (GSGSG). Forcomparison, a polymer composed of monomers bearing a single guanidiniummoiety was prepared as a graft-through analogue of polymers prepared viathe graft-to technique employed in other studies. These graft-toguanidinium-containing polymers are the only other cell penetratingpolymers prepared by ROMP techniques. The graft-to guanidinium polymerprepared displayed poor solubility as a homopolymer and was thereforeprepared as a block copolymer with a water-solubilizing OEG monomer(FIG. 1B, where R₁=OEG and R₂=guanidinium). After polymerization, thepolymers were characterized by size-exclusion chromatography withmultiangle light scattering (SEC-MALS) to ascertain degree ofpolymerization (DP) and molecular weight distribution (dispersity orM_(w)/M_(n)) (Table 1). Good agreement between the obtained DP and thetheoretical DP based on the initial monomer-to-initiator ratio([M]₀/[I]₀) was observed. Further, all dispersities were less than 1.11,indicating the expected narrow molecular weight distributions.

A chromatogram showing characterization of OEG polymer by SEC-MALS (sizeexclusion chromatography, multi-angle static light scattering) wasobtained, including SLS and. Solid colored line indicates SLS, and blackdotted line indicates the refractive index (RI). SLS data was onlytabulated for peaks that have a corresponding RI signal. Thechromatogram is noisy in the SLS component because the polymer runscoincident with the solvent front due to its low molecular weight(˜3500). A chromatogram showing characterization of GSGSG polymer bySEC-MALS was obtained. A chromatogram showing characterization of Arg8polymer by SEC-MALS was obtained. A chromatogram showingcharacterization of OEG-b-Guanidinium polymer, first “m” block (OEG) bySEC-MALS was obtained. A chromatogram showing characterization ofOEG-b-Guanidinium polymer, second “n” block (guanidinium) by SEC-MALSwas obtained. A chromatogram showing characterization of phenyl-b-GSGSGpolymer, first “m” block (Phenyl) by SEC-MALS was obtained. Achromatogram showing characterization of phenyl-b-GSGSG polymer, second“n” block (GSGSG) (SEQ ID NO: 1) by SEC-MALS was obtained. Achromatogram showing characterization phenyl-b-Tat polymer, first “m”block (Phenyl) by SEC-MALS was obtained. A chromatogram showingcharacterization phenyl-b-Tat polymer, second “n” block (Phenyl) bySEC-MALS was obtained. Solid colored line indicates SLS, and blackdotted line indicates the refractive index (RI). Red traces representhomopolymers or the first block of a block copolymer while the secondblock of block polymers are shown in blue. SLS data is only tabulatedfor peaks that have a corresponding RI signal.

A plot showing side and forward scattering of the region gated for eachflow cytometry experiment was obtained. The percentage given is thepercentage of cells that fall within the gated region (10,000 eventstotal). Experiment was performed in triplicate on separate cultures ofHeLa cells, and the data for one recording is given.

A histogram showing fluorescence intensity of the gated region for thevehicle control was obtained. Y-axis is number of cells and x-axis isfluorescence. Experiment was performed in triplicate on separatecultures of HeLa cells, and the data for one recording is given.

TABLE 7 Characterization of cell penetrating polymer and controls^(a)Block m Block n Polymer M_(n) ^(a) M_(w)/M_(n) ^(b) DP^(c) M_(n) ^(a)M_(w)/M_(n) ^(b) DP^(d) OEG (10 mer) 3,600 1.03 10 (10) — — — OEG (25mer) 8,800 1.07 25 (25) — — — GSGSG 5,700 1.05 10 (12) — — — Tat 8,600n/a 5 (6) — — — Arg8 36,000 1.08 8 (6) — — — PEG- 8,300 1.02 23 (15)12,000 1.07 8 (12) Guanidium Phenyl-GSGSG 14,000 1.02 54 (70) 18,0001.02 8 (12) Phenyl-Tat 13,000 1.01 52 (70) 22,000 1.11 5 (6)  *Block mand n refer to the first and second block to be polymerized as shown inFIGS. 1A-1B. ^(T)Each polymer is named according to identity of themonomer polymerized as drawn in FIGS. 1A-1B. Block co-polymers arelisted with block m first and block n second, separated by a hyphen.Further annotation is as follows: ^(a)The number average molecularweight, ^(b)the dispersity of each block with theoretical values basedbased on the amount of material used given in parentheses, c, ^(d)degreeof polymerization or m (c) and n (d) as denoted in FIG. 1B. All datawere obtained by SEC-MALS, except for those describing the Tat polymer,which did not elute on the SEC column and was instead characterized in acuvette by static light scattering. Without the SEC component, noinformation on the molecular weight distribution of this polymer wasobtained. However, the amphipilic polymer that contains Tat eluted wellon SEC and yielded close to the predicted DP in low dispersity.

The Tat peptide-containing homopolymer, lacking side-chain protectinggroups, performed poorly on SEC-MALS, presumably due to unfavorableinteractions of the peptide with the size exclusion column. Therefore,no information on the molecular weight distribution of these polymerswas obtained, but a molecular weight determination was achieved bymeasuring the bulk light scattering of the solution in a cuvette(without the size exclusion column); and complete consumption of the Tatmonomer after polymerization was verified by ¹H NMR. An ¹H NMR spectrumof NorAha-GSGSG polymerization of monomers. Initial spectrum was takenprior to addition of the initiator. Resonance at δ 6.32 ppm correspondsto the norbornene olefin protons of the monomer. Subsequent spectrum wasrecorded at the end of the polymerization (t=3 hr) and verifies completeconsumption of the monomer (no resonance at δ 6.32 ppm). The newresonance at ˜δ 5.5-6 ppm corresponds to the cis-trans olefin protons ofthe polymerized material. An ¹H NMR spectrum NorGuanidiniumpolymerization of monomers was obtained. Initial spectrum was takenprior to addition of the initiator. Resonance at δ 6.32 ppm correspondsto the norbornene olefin protons of the monomer. Subsequent spectrum wasrecorded at the end of the polymerization (t=3 hr) and verifies completeconsumption of the monomer (no resonance at δ 6.32 ppm). The newresonance at ˜δ 5.5-6 ppm corresponds to the cis-trans olefin protons ofthe polymerized material. An ¹H NMR spectrum of NorAha-Tatpolymerization of monomers was obtained. Initial spectrum was takenprior to addition of the initiator. Resonance at δ 6.32 ppm correspondsto the norbornene olefin protons of the monomer. Subsequent spectrum wasrecorded at the end of the polymerization (t=3 hr) and verifies completeconsumption of the monomer (no resonance at δ 6.32 ppm). The newresonance at ˜δ 5.5-6 ppm corresponds to the cis-trans olefin protons ofthe polymerized material.

Given the complexity of the Tat and Arg8 peptide-containing polymers(i.e., multiple charged and nucleophilic side chains), it wasinvestigated whether ROMP of these materials proceeds in a livingfashion, in order to ensure that well-defined and well-orderedstructures, devoid of cross-metathesis or premature termination, couldregularly be accessed by this strategy. Confirming the living nature ofthe polymerization, a plot of M_(n) (obtained by SEC-MALS) vs [M]₀/[I]₀for the Arg8 monomer yields a linear fit for [M]₀/[I]₀ less than 9(Table 2). At larger [M]₀/[I]₀ ratios, propagation ceased, presumablydue to steric hindrance encountered from assembling multiple copies ofthe long, side-chain protected peptide sequence, whose molecular weightas a monomer is 3.5 kDa. A similar plot was obtained from data gatheredfor polymerization of the Tat polymer, collected by static lightscattering (SLS) in a cuvette (and Table 2). Therefore, both CPPmonomers were polymerized in a living fashion to a DP of <9, despite thecomplexity and functionality of their side chains, making this anexceptionally convenient strategy for predictably generating polymericarchitectures from peptide monomers.

Plots correlating the number-average molecular weight (Mn) with theinitial monomer-to-catalyst ratio ([M₀/I₀] for the polymerization of theArg8 monomer were calculated. Linear fits are indicative of a livingpolymerization. Propagation ceased after the polymerization of ˜9monomers.

Plots correlating the number-average molecular weight (Mn) with theinitial monomer-to-catalyst ratio ([M₀/I₀] for the polymerization of theTat monomer were calculated. Linear fits are indicative of a livingpolymerization. Propagation ceased after the polymerization of ˜9monomers.

TABLE 8 Characterization of the polymerization of the Tat and Arg8monomers at multiple initial monomer-to-catalyst ratios TatPolymerization Arg8 Polymerization M_(w)/ [M]₀:[I]₀ ^(a) M_(n) ^(b)DP^(c) M_(w)/M_(n) ^(d) [M]₀:[I]₀ ^(a) M_(n) ^(b) DP^(c) M_(n) ^(d) 511000 3 1.05 5 6400 4 n/a 10 20000 6 1.05 10 9600 5 n/a 15 30000 9 1.0315 15000 8 n/a 20 27000 8 1.05 20 14000 8 n/a 40 31000 9 1.07 40 16000 9n/a 60 23000 6 1.08 60 15000 8 n/a *The polymers listed are allhomopolymers as shown in FIG. 1A. Annotation is described as: ^(a)theinitial monomer-to-catalyst ratio used, ^(b)the number average molecularweight obtained, ^(c)the degree of polymerization obtained ^(d)thedispersity of the polymerization. All data for the Arg8 monomerpolymerization were collected by SEC-MALS. Data for the Tat monomerpolymerization were obtained in a cuvette via static light scattering,so no information on the dispersity of the Tat polymers was obtained.

Proteolytic Resistance and Bioactivity of Large Assemblies ofPeptide-Containing Polymers

In addition to exploring the activity of a single polymer chain, theproteolytic resistance and bioactivity of large assemblies ofpeptide-containing polymers were examined. Although it was predictedthat nanoscale assemblies of multiple peptide-polymers would be largeenough to avoid renal clearance thresholds in future applications thatmight otherwise prevent long circulation times of peptides or lowermolecular weight polymers, it was unclear whether these large assemblieswould resist proteolysis, or enter cells. To generate nano-particles,amphiphilic polymers of two peptides were prepared: Tat and a GSGSGcontrol peptide (FIG. 1B). The design of these amphiphiles was such thatthe phenyl-modified norbornene monomers operated as hydrophobic moietiesto drive self-assembly of the amphiphiles into micellar nano-particlescontaining many copies of polymers. To prepare these amphiphiles, ahydrophobic monomer was polymerized to completion prior to addition ofthe peptide monomer to the living polymer (FIG. 1B, where R₁ is phenyland R₂ is GSGSG or Tat). Self-assembly of these amphiphilic polymersinto a nanoscale structure was then accomplished by slow dialysis of thematerial from an organic co-solvent, in which the amphiphile wascompletely dissolved (DMF), into a selective solvent, in which only thepeptide brush is soluble (aqueous phosphate-buffered saline, PBS). Theamphiphilic polymers of Tat and GSGSG were found, by DLS and TEM, toform spherical micelles of ˜10-50 nm diameter (FIGS. 2A and 2B). DLSdata for the Tat and GSGSG particles indicate maximum intensity at about80 nm radius.

Example 2: Cellular Uptake in HeLa Cells by Flow Cytometry and Live-CellConfocal Microscopy

Cell Culture Assay Methods

HeLa cells were purchased from ATCC (CCL-2). Cells were cultured at 37°C. under 5% CO₂ in phenol red-containing Dulbecco's Modified EagleMedium (DMEM; Gibco Life Tech., cat. #11960-044) supplemented with 10%fetal bovine serum (Omega Scientific, cat. #FB02) and with 1×concentrations of nonessential amino acids (Gibco Life Tech., cat.#11140-050) sodium pyruvate (Gibco Life Tech., cat. #11360-070),L-glutamine (Gibco Life Tech., cat. #35050-061), and the antibioticspenicillin/streptomycin (Corning Cellgro, cat. #30-002-C1). Cells weregrown in T75 culture flasks and subcultured at ˜75-80% confluency (every˜3-4 days). An image showing the live-cell confocal microscopy images ofthe GSGSGKK polymer (DP˜60) was obtained. All images are the averageintensity from six consecutive 1 μm Z-slices using a 40× objective.Cells were treated with 2.5 μM of the material (with respect tofluorophore). GSGSGKK polymer has a peptide m of ˜60 and the KLA polymeris m˜10. Each polymer that contains Lys or Arg shows a mixture ofdiffuse and punctate fluorescence, indicating that the material residesin the cytosol and in cellular compartments, respectively. An imageshowing the live-cell confocal microscopy images of the GSGSGRR polymer(DP˜60) was obtained. All images are the average intensity from sixconsecutive 1 μm Z-slices using a 40× objective. Cells were treated with2.5 μM of the material (with respect to fluorophore). GSGSGRR polymerhas a peptide m of ˜60 and the KLA polymer is m˜10. An image showing thelive-cell confocal microscopy images of the KLA polymer (DP˜10) wasobtained. All images are the average intensity from six consecutive 1 μmZ-slices using a 40× objective. Cells were treated with 2.5 μM of thematerial (with respect to fluorophore). KLA polymer has a peptide m of˜10 and the KLA polymer is m˜10.

Flow Cytometry Methods

HeLa cells were plated at a density of 90,000 cells per well of a24-well plate 18 h prior to treatment. Materials dissolved in Dulbecco'sPhosphate Buffered Saline (DPBS without Ca²⁺ or Mg²⁺; Corning Cellgro,cat. #21-031-CM) at 10× the desired concentration were added to thewells, and the plates were incubated for 30 min at 37° C. The medium wasthen removed, and the cells were washed twice with DPBS and thenincubated three times for 5 min with heparin (0.5 mg/mL in DPBS;Affymetrix, cat. #16920), and finally rinsed again with DPBS. The cellswere then trypsinized (0.25% trypsin; Gibco Life Tech., cat. #15090-046)for 10 min, cold medium was added, and the cells were transferred toEppendorfs, centrifuged to pellets and then resuspended in a minimalamount of cold DPBS. Fluorescence activated cell sorting data (10000events on three separate cultures) was acquired on an Accuri C6 flowcytometer set to default “3 blue 1 red” configuration with standardoptics and slow fluidics (14 μL/min). For proteolysis studies, theindicated concentration of Tat peptide, homopolymer or particle waspretreated with 1 μM of trypsin, chymotrypsin or Pronase for 20 min inDPBS, after which the protease was heat denatured for 15 min at 65° C.The cells were then incubated, prepared and analyzed by flow cytometryas described above. For mechanistic studies, cells were preincubatedwith the indicated compound for 30 min at 37° C. prior to addition ofthe cell-penetrating material. The following concentrations were used:80 μM dynasore (Enzo Life Sciences, cat. #270-502-M05) and 9.5 mM MJ3CD(Fischer Scientific, cat. #AC377110050). For studies at reducedtemperature, cells were incubated at 4° C. for 30 min prior to andduring the incubation with the compound of interest. All subsequentwashes and manipulations were also done with ice-cooled media and othermaterials. Data is reported as the normalized mean fluorescence, whichare the mean fluorescence yielded by the material/the mean fluorescencefrom the vehicle control.

Live-Cell Confocal Microscopy Methods

HeLa cells were seeded on glass-bottom 24-well plates at a cell densityof 90,000 cells per well 18 h prior to treatment. The medium was removedand then replaced with medium lacking phenol red (Gibco Life Tech.,cat#31053-028) to minimize background fluorescence. Materials dissolvedin DPBS (at 10× the desired concentration) were added to the wells andthe plates were incubated for 30 min at 37° C. The washing procedureused in the flow cytometry experiments (2×DPBS, 3×heparin for 5 min,1×DPBS) was followed here. Following removal of the final DPBS rinsate,fresh media (phenol red-free) was added to each well. Live cells wereimaged on an Olympus FV1000 confocal microscope. For proteolysisstudies, the indicated concentration of Tat peptide, homopolymer andparticle were pretreated with 1 μM of trypsin, chymotrypsin or Pronasefor 20 min in DPBS, after which the protease was heat denatured for 15min at 65° C. The cells were then incubated, prepared and analyzed byconfocal microscopy as described above.

Cellular Uptake of Peptide Controls, Polymer and Nanoparticles

Fluorescence-based in vitro assays were performed in HeLa cells tocompare the cellular uptake of the peptide controls, polymers, andnanoparticles. The goal was to determine whether polymerization of theCPPs had an impact on their ability to facilitate cellular entry or ontheir mechanisms of cellular uptake. In these studies, flow cytometrywas used to quantify the amount of cellular uptake, and live-cellconfocal microscopy was used to verify internalization and examine thelocalization of the internalized material.

In flow cytometry experiments, relative to the vehicle control (PBS),all CPP-containing peptide polymers and particles gave robustfluorescent counts, approximately 2-fold higher than those of thepeptides alone (FIG. 3 and FIGS. 5A-5B). These data verify that CPPsmaintain or have enhanced function when incorporated into brush polymersor larger polymeric assemblies. The Tat and Arg8 polymers gave responsessimilar to those of the guanidinium polymer, which is an analogue of theonly other cell-penetrating ROMP polymer reported to date. A graphdepicting flow cytometry data for fluorescein was obtained; recordingswere gated to the vehicle control (PBS). A graph depicting flowcytometry data for the OEG polymer was obtained; recordings were gatedto the vehicle control (PBS). A histogram showing any shifts influorescence of fluorescein (in green) relative to the vehicle controlwas obtained. A histogram showing any shifts in fluorescence of the OEGpolymer relative to the vehicle control was obtained. A graph depictingflow cytometry data for the GSGSG (SEQ ID NO: 1) peptide was obtained;recordings were gated to the vehicle control (PBS). The observedpercentage (50.2) refers to the cells within the gated region for eachsample (10,000 events total). A graph depicting flow cytometry data forthe GSGSG (SEQ ID NO: 1) polymer was obtained; recordings were gated tothe vehicle control (PBS). The observed percentage (35.9) refers to thecells within the gated region for each sample (10,000 events total). Agraph depicting flow cytometry data for the GSGSG (SEQ ID NO: 1)particle was obtained; recordings were gated to the vehicle control(PBS). The observed percentage (37.7) refers to the cells within thegated region for each sample (10,000 events total). A histogram showingany shifts in fluorescence of GSGSG (SEQ ID NO: 1) peptide relative tothe vehicle control was constructed. A histogram showing any shifts influorescence of the GSGSG (SEQ ID NO: 1) polymer relative to the vehiclecontrol was constructed. A histogram showing any shifts in fluorescenceof the GSGSG (SEQ ID NO: 1) particle relative to the vehicle control wasconstructed. A graph depicting flow cytometry data for the Tat Peptidewas obtained; recordings were gated to the vehicle control (PBS). Theobserved percentage (58.2) refers to the cells within the gated regionfor each sample (10,000 events total). A graph depicting flow cytometrydata for the Tat Polymer was obtained; recordings were gated to thevehicle control (PBS). The observed percentage (41.2) refers to thecells within the gated region for each sample (10,000 events total). Agraph depicting flow cytometry data for the Tat Particle was obtained;recordings were gated to the vehicle control (PBS). The observedpercentage (47.9) refers to the cells within the gated region for eachsample (10,000 events total). A histogram showing any shifts influorescence of the Tat Peptide relative to the vehicle control wasconstructed. A histogram showing any shifts in fluorescence of the TatPolymer relative to the vehicle control was constructed. A histogramshowing any shifts in fluorescence of the Tat Particle relative to thevehicle control was constructed. A graph depicting flow cytometry datafor the Arg8 Peptide was obtained; recordings were gated to the vehiclecontrol (PBS). The observed percentage (71.8) refers to the cells withinthe gated region for each sample (10,000 events total). A graphdepicting flow cytometry data for the Arg8 Polymer was obtained;recordings were gated to the vehicle control (PBS). The observedpercentage (32.7) refers to the cells within the gated region for eachsample (10,000 events total). A graph depicting flow cytometry data forthe Guanidinium Polymer was obtained; recordings were gated to thevehicle control (PBS). The observed percentage (76.2) refers to thecells within the gated region for each sample (10,000 events total). Ahistogram showing any shifts in fluorescence of the Arg8 Peptiderelative to the vehicle control was constructed. A histogram showing anyshifts in fluorescence of the Arg8 Polymer relative to the vehiclecontrol was constructed. A histogram showing any shifts in fluorescenceof the Guanidinium Polymer relative to the vehicle control wasconstructed.

To probe whether the cellular uptake of the polymerized materials wasdue to the peptide amino acid sequence and not the polymer backboneitself, or the result of the arrangement of any peptide into a brushpolymer, the uptake of polymeric materials containing an OEG brush and aGSGSG (SEQ ID NO: 1) brush were investigated, both of which do not entercells as their monomer units. The control materials showed negligiblefluorescence signals (less than a 2-fold increase in fluorescencerelative to vehicle), similar to the small molecule fluorescein tagitself. Therefore, these data indicate that the amino acid sequences ofTat and Arg8 drive the internalization of the polymers.

Compartmentalization of Fluorescence within Cytoplasm

To confirm that the fluorescence observed in the initial studies residedwithin the cytoplasm, rather than on the cell's external surface. Tothis end, live-cell confocal microscopy was performed, because fixationof cells by formaldehyde, methanol or other agents, can cause artifactsdue to the release of fluorescently labeled materials entrapped inendosomes. In particular, Z-stack analyses was performed at 1 μm stepsizes on live cells treated with each peptide-based material. Acrossmultiple Z-slices for cells treated with all Tat-, Arg8- andguanidinium-containing materials, at the same concentration used in flowcytometry experiments, a combination of punctate and diffusefluorescence was observed, indicative of compartmentalized and cytosoliclocalization, respectively. By contrast, no fluorescence was seen forany of the negative controls (GSGSG and OEG polymers), which did notcontain cationic moieties and did not penetrate cells in any detectablemanner. Images showing the consecutive Z-stack slices that were averagedtogether to yield the averaged image of the Tat peptide were obtained.Slices were acquired every 1 μm. Punctate and diffuse fluorescence areseen in each slice, indicating that the Tat peptide has permeated thecell and does not just reside at the cell membrane.

Polymerization Toxicity to Cells

Polymerization did not render the peptides toxic to cells. The viabilityof cells treated with the Tat and GSGSG (SEQ ID NO: 1) peptide, polymerand nanoparticles was assayed via the CellTiter-Blue assay. Whencompared to the vehicle control, HeLa cells treated with allformulations of the materials at 5 μM, twice the concentration used inthe uptake studies described above, remained >92% viable for 48 h (FIG.11).

Example 3: The Mechanism of Cellular Uptake

Cell Viability Assay Methods

The CellTiter-Blue assay (Promega, cat. #G8081) measures the reductionof resazurin to resorufin via fluorescence. HeLa cells were plated at adensity of 4,000 cells per well of a 96-well plate 18 h prior totreatment. Materials dissolved in DPBS at 5 μM were added to the wellsalong with a 10% DMSO positive control. Cells were incubated for 48 h at37° C. The medium was removed and 80 μL of fresh media lacking phenolred was added. To this was added 20 μL of the CellTiter-Blue reagent andthe cells were then incubated for 2 h prior to measuring fluorescence ina plate reader using 560 nm excitation and 590 nm emission. Thefluorescence measurements were corrected for background fluorescencefrom the CellTiter-Blue reagent by subtracting the fluorescence readingof wells treated with the reagent in the absence of cells. Fluorescencevalues were then referenced as a percentage of the value obtained forthe PBS vehicle control.

Internalization Routes of CPPs Via Thermal Inhibition andPharmacological Compounds

Much debate in the literature resides over the mechanism of entry ofCPPs. However, it is generally agreed that the cellular uptake of thesematerials requires association with anionic species at the cell membrane(i.e., sulfated proteoglycans or phospholipid polar headgroups) followedby internalization via endocytosis or membrane disruption. Toinvestigate whether the monomeric, polymeric, and nanoparticleformulations of the CPPs follow similar internalization routes, cellswere subjected to thermal inhibition and common pharmacologicalcompounds that disrupt different aspects of membrane trafficking andendocytosis.

First, membrane trafficking was arrested by reducing the incubationtemperature to 4° C. This resulted in a dramatic decrease in thefluorescent signals for the Tat, Arg8, and guanidinium polymers andnanoparticles by flow cytometry, but had no influence on the values fromthe GSGSG controls (FIG. 5A). Similar effects were seen with aninhibitor of dynamin-dependent endocytosis (dynasore) and also withmethyl-β-cyclodextrin (MβCD), an agent known to remove membranecholesterol, and thereby alters the fluidity of the membrane. Eachcondition resulted in no change in the fluorescence values obtained forthe GSGSG controls, which was consistent with the notion that theseuncharged materials do not internalize. The polymers containingguanidinium, Tat and Arg8 side chains all showed uptake, as was expectedfor these modes of inhibition. Note that, in all cases, pharmacologicaland thermal inhibition exhibited only a small effect on the flowcytometry readings of the Tat and Arg8 peptides. Together, these resultsindicate that cell penetration of the peptides (individual CPPs) is due,in part; to membrane disruption or endocytotic processes and that thesemechanisms of entry are maintained or enhanced upon polymerization.

Effect of Fetal Bovine Serum (FBS) Components on Cellular Entry

To verify that the wide range of components found in fetal bovine serum(FBS) did not play a role in facilitating or inhibiting cellular entryof the materials, experiments were also performed in FBS-free media. Nosignificant difference in mean fluorescence from any material wasobserved by flow cytometry in the presence or absence of FBS, indicatingthat FBS components, such as growth factors, lipids, hormones, etc., didnot influence uptake of these materials.

A bar graph showing flow cytometry data for materials incubated with andwithout fetal bovine serum was obtained. No significant differences invalues were obtained, indicating that serum components do not affectcellular uptake of the materials. A standard curve used to determineconcentration of fluorescein-containing materials was obtained. Data wasrecorded and fit for the small molecule 5/6-carboxyfluorescein andconfirmed for use with the fluorescein-Tat polymers and particles.Absorbance was at 492 nm. A standard curve for peak area of untreatedTat peptide-99 μL injections was obtained. A standard curve for peakarea of untreated Tat peptide-20 μL injections was obtained. A standardcurve for peak area of untreated Tat polymer-99 μL injections wasobtained. A standard curve for peak area of untreated Tat particle-15 μLinjections was obtained. A chromatogram showing Tat peptide at 2.5 μMwith chymotrypsin before and after (dotted black line) treatment with 1μM protease was obtained. A chromatogram showing Tat peptide at 2.5 μMwith trypsin before and after treatment with 1 μM protease was obtained.A chromatogram showing Tat peptide at 2.5 μM with pronase before andafter treatment with 1 μM protease was obtained. A chromatogram showingTat peptide at 12.5 μM with chymotrypsin before and after treatment with1μM protease was obtained. A chromatogram showing Tat peptide at 12.5 μMwith trypsin before and after treatment with 1 μM protease was obtained.A chromatogram showing Tat peptide at 12.5 μM with pronase before andafter treatment with 1 μM protease was obtained. A chromatogram showingTat polymer at 2.5 μM with chymotrypsin before and after treatment with1 μM protease was obtained. A chromatogram showing Tat polymer at 2.5 μMwith trypsin before and after treatment with 1 μM protease was obtained.A chromatogram showing Tat polymer at 2.5 μM with pronase before andafter treatment with 1 μM protease was obtained. A chromatogram showingTat particle at 2.5 μM with chymotrypsin before and after treatment with1 μM protease was obtained. A chromatogram showing Tat particle at 2.5μM with trypsin before and after treatment with 1 μM protease wasobtained. A chromatogram showing Tat particle at 2.5 μM with pronasebefore and after treatment with 1 μM protease was obtained. A bar graphshowing the time course of the treatment of the TAT polymer (2.5 μM)with chymotrypsin (1μM) was obtained. The peptide was cleaved to lessthan 10% of the starting concentration of material after 20 minutes ofincubation with chymotrypsin. The functional impact of proteolytictreatment is measured by flow cytometry while the percentage of intactpeptide remaining after incubation was assayed by RP-HPLC.

An ¹H NMR time course spectra for the polymerization of R control wasobtained. Polymers were polymerized to a DP (m) of ˜10. Initial spectrawere taken at t=0 and subsequent spectra were taken at t=3 hr. Adisappearance was observed of the resonance at δ=6.32 ppm correspondingto the olefin protons of the monomer and the coincident appearance ofresonances at δ=5.5-6 ppm, which correspond to the cis and trans olefinprotons of the polymer backbone. An ¹H NMR time course spectra for thepolymerization of GSGSGR was obtained. Polymers were polymerized to a DP(m) of ˜10. Initial spectra were taken at t=0 and subsequent spectrawere taken at t=3 hr. A disappearance was observed of the resonance atδ=6.32 ppm corresponding to the olefin protons of the monomer and thecoincident appearance of resonances at δ=5.5-6 ppm, which correspond tothe cis and trans olefin protons of the polymer backbone. An ¹H NMR timecourse spectra for the polymerization of GSGSGR was obtained. Polymersare polymerized to a DP (m) of ˜10. Initial spectra were taken at t=0and subsequent spectra were taken at t=3 hr. A disappearance was observed of the resonance at δ=6.32 ppm corresponding to the olefinprotons of the monomer and the coincident appearance of resonances atδ=5.5-6 ppm, which correspond to the cis and trans olefin protons of thepolymer backbone. An ¹H NMR time course spectra for the polymerizationof RRGSGSG was obtained. Polymers are polymerized to a DP (m) of ˜10.Initial spectra were taken at t=0 and subsequent spectra were taken att=3 hr. A disappearance was observed of the resonance at δ=6.32 ppmcorresponding to the olefin protons of the monomer and the coincidentappearance of resonances at δ=5.5-6 ppm, which correspond to the cis andtrans olefin protons of the polymer backbone. An ¹H NMR time coursespectra for the polymerization of GSGSGRR was obtained. Polymers werepolymerized to a DP (m) of ˜10. Initial spectra were taken at t=0 andsubsequent spectra were taken at t=3 hr. A disappearance was observed ofthe resonance at δ=6.32 ppm corresponding to the olefin protons of themonomer and the coincident appearance of resonances at δ=5.5-6 ppm,which correspond to the cis and trans olefin protons of the polymerbackbone. An ¹H NMR time course spectra for the polymerization of GSGSGKwas obtained. Polymers were polymerized to a DP (m) of ˜10. Initialspectra were taken at t=0 and subsequent spectra were taken at t=3 hr. Adisappearance was observed of the resonance at δ=6.32 ppm correspondingto the olefin protons of the monomer and the coincident appearance ofresonances at δ=5.5-6 ppm, which correspond to the cis and trans olefinprotons of the polymer backbone. An ¹H NMR time course spectra for thepolymerization of GSGSGKK was obtained. Polymers were polymerized to aDP (m) of ˜10. Initial spectra were taken at t=0 and subsequent spectrawere taken at t=3 hr. A disappearance was observed of the resonance atδ=6.32 ppm corresponding to the olefin protons of the monomer and thecoincident appearance of resonances at δ=5.5-6 ppm, which correspond tothe cis and trans olefin protons of the polymer backbone. An ¹H NMR timecourse spectra for the polymerization of KLA (full length) was obtained.Polymers were polymerized to a DP (m) of ˜10. Initial spectra were takenat t=0 and subsequent spectra were taken at t=3 hr. A disappearance wasobserved of the resonance at δ=6.32 ppm corresponding to the olefinprotons of the monomer and the coincident appearance of resonances atδ=5.5-6 ppm, which correspond to the cis and trans olefin protons of thepolymer backbone. An ¹H NMR time course spectra for the polymerizationof KLA (fragment) was obtained. Polymers were polymerized to a DP (m) of˜10. Initial spectra were taken at t=0 and subsequent spectra were takenat t=3 hr. A disappearance was observed of the resonance at δ=6.32 ppmcorresponding to the olefin protons of the monomer and the coincidentappearance of resonances at δ=5.5-6 ppm, which correspond to the cis andtrans olefin protons of the polymer backbone.

Effect of Peptide Concentration on Cellular Uptake

To examine how the concentration of the Tat peptide-containing materialsimpacted their cellular uptake, flow cytometry experiments wereperformed at several concentrations of material. The concentration inthese experiments was with respect to the fluorophore, where there wasone fluorophore per peptide or polymer, but many copies of fluorophoreper particle. In general, the peptide was less competent in cellpenetration than the polymer or particle formulations, with cellularuptake of the peptide nearly abolished at 1.25 μM. This is in contrastto the polymer and nanoparticles that were still taken up by cells atconcentrations as low as 0.5 μM (FIG. 5B).

Effect on Number of Guanidiniums for Cell Penetration

Studies on linear peptides have shown that 8-16 guanidiniums are optimalfor cell penetration, with activity dramatically decreasing when over 16guanidiniums were used. Likewise, polynorbornyl polymers bearingguanidi-nium moieties showed decreased internalization when 25guanidiniums were incorporated, compared to when 10 were employed.Efficient penetration for the Tat side chain 5-mer homopolymer andnanoparticle, which contains at least 30 guanindinium units (FIG. 5B)was observed. In fact, the polymer penetrated cells as efficiently(within a factor of 2 fluorescence counts) as the relevant peptideanalogue, even in scenarios in which 5-fold fewer fluorophores werepresent to achieve the same effective concentration of peptide (such as2.5 M Tat polymer and 12.5 M Tat peptide). These data indicate that thearrangement of the brush polymer may aid in the cellular uptakemechanism, which could require assembly of multiple CPPs for propertransport across the membrane. Indeed, oligomerization of CPPs into“carpet” bundles and direct transportation of these bundles across themembrane has been proposed for many years as the so-called “carpetmechanism”. Alternatively, efficient cellular entry could be due totangled pendant peptide chains presenting a lower effective number ofcharged residues to the cell membrane.

Example 4: Evaluation of Proteolysis of Cell Penetrating Peptides,Polymers and Nanoparticles

RP-HPLC Analysis of CPP Proteolysis Methods

The extent of proteolytic degradation of the Tat peptide, polymer andparticle by trypsin (Gibco Life Tech., cat. #15090-046), a-chymotrypsin(Fisher Scientific, cat. #ICN1522722) and Pronase (Roche, cat.#10165921001) was assessed by comparison of chromatograms in RP-HPLC. Inthese experiments, the material at the indicated concentration wasincubated with each protease (at 1 μM) for 20 min, and then the enzymeswere heat denatured at 65° C. for 15 min, and the solution wasimmediately injected onto an analytical RP-HPLC. Given that treatmentwith each protease gives multiple fragments of the Tat sequence, astandard curve for each starting material was prepared to assess thepercentage of intact material remaining after proteolytic digestion.Note that the standard curves for the polymer and particle will bebiased due to the fact that after cleavage, the polymer backbone andfluorophore should remain intact, and will comprise part of the measuredpeak area. Nevertheless, no new peaks were seen in the chromatograms ofthe polymer or particle post enzyme treatment, suggesting that thesematerials are not susceptible to cleavage by the proteases.

Resistance to Proteolysis in Cell-Penetrating Materials

The resistance to proteolysis was assessed in the cell-penetratingmaterials. The proteolytic cleavage of materials containing the Tatpeptide was emphasized, given that it had a more diverse amino acidsequence than the Arg8 peptide and would therefore have more uniquecleavage sites.

Tat-containing materials, at the same concentration used in flowcytometery and confocal microscopy studies described above (2.5 M), werechallenged for 20 min with various proteases at high enzymeconcentration (˜1 M) prior to determining the extent of proteolyticcleavage and residual bioactivity. Such activity was assayed by threeseparate methods: reverse-phase high-performance liquid chromatography(RP-HPLC), flow cytometry, and confocal microscopy. In these assays,RP-HPLC was used to determine the degree to which proteolytic treatmentdegrades the integrity of the peptide as a monomer or as part of apolymeric formulation. The bioactivity of enzymatically digestedmaterials was then assessed in cellular assays by both flow cytometryand confocal microscopy. To determine whether the location of thepeptide cleavage site(s) affects the sensitivity of the peptide toenzymatic cleavage, several different proteases were tested: trypsin (7predicted cleavage sites), chymotrypsin (2 predicted cleavage sites),and the protease cocktail Pronase, which had the potential to digest thepeptide backbone at every amino acid position.

RP-HPLC was used to assess the percent of intact material followingenzymatic digestion (FIG. 6A). Standard curves were generated thatcompared peak areas of the uncleaved Tat peptide, polymer, ornanoparticle at an appropriate concentration range, such that theconcentration of material remaining after incubation with enzyme couldbe estimated. The absorbance measurements of the intact polymer wereaffected by the norbornyl polymer backbone, the phenyl coblock, and thefluorescein end-label, which were still present after proteolyticdigestion. In these assays, no differences in the peak area or retentiontime of the polymer or particle were observed after treatment with anyof the proteases tested and the RP-HPLC chromatograms were identicalwith and without enzyme treatment (i.e. no new peaks formed in thechromatogram), indicating that the Tat polymers and particles wereresistant to proteolysis. By contrast, complete consumption of the Tatpeptide was detected, along with the appearance of new peaks in theRP-HPLC chromatograms. Correspondingly, the mean fluorescence counts ofthe polymer or particle measured by flow cytometry were largelyunaffected by protease treatment (presumably since the peptide chainshave not been digested). However, proteolytic digestion of the Tatpeptide diminished the intensity of the fluorescence signal to less than10% of the value obtained prior to enzymatic digestion (FIG. 7B). Thesame trends were observed by both RP-HPLC and flow cytometry when thepeptide concentration was kept uniform (12.5 M peptide, 2.5 M polymer)to normalize the number of potential cleavage events. This differencewas substantial given that the peptides had 5 times the number offluorescein (1 per peptide) equivalents per peptide than the polymer (1per 5 peptides). Furthermore, a time-course plot of RP-HPLC and flowcytometry data revealed that the Tat polymer was stable to chymotrypsintreatment over 14 h and the material retained the ability to enter cellsafter incubation with the enzyme.

Confocal microscopy was used to verify trends observed by RP-HPLC andflow cytometry. In these experiments, the Tat peptide, Tat polymer, andTat nanoparticle were pretreated with chymotrypsin (under identicalconditions as used in the flow cytometry and RP-HPLC assays) prior toincubation with HeLa cells. These cells were then imaged by live-cellconfocal microscopy alongside cells incubated with the same materialsthat had not been subjected to the enzyme pretreatment. A dramaticcomparison emerged in which cells treated with protease-digested Tatpeptides showed minimal fluorescence relative to those treated withundigested peptides (FIG. 7C). In contrast, the Tat polymer andparticles gave identical fluorescence images with or without enzymetreatment.

Example 5: Assessment of Approach Utilizing Two Additional ProteaseSubstrates Polymerization of Thrombin Substrate Methods

Polymerizations were carried out in a glovebox under a N₂ (g)atmosphere. To generate the polymers containing the thrombin peptidesequence, the monomer (0.007 mmol, 10 equiv, 23 mM for DP=10; 0.013mmol, 20 equiv, 45 mM for DP=20; 0.021 mmol, 30 equiv, 70 mM for DP=30)was mixed with the catalyst (0.0007 mmol, 1 equiv, 2.3 mM) in DMF-d₇(0.3 mL) and monitored by ¹H NMR to confirm complete consumption of themonomer and to determine the time period required to reach completion.Upon completion, the polymers were quenched with ethyl vinyl ether for10 min, and then precipitated with cold ether and dried under a vacuum.The resulting polymers were directly characterized by SEC-MALS.

RP-HPLC Analysis of Thrombin Proteolysis Methods

The extent of proteolytic degradation of the thrombin peptide polymersby thrombin (Sigma, cat. #T6884-100UN) was assessed by comparison ofchromatograms in reverse-phase HPLC. In these experiments, the monomerand polymers were dissolved in PBS buffer (2.2 mM with respect topeptide). Thrombin (10 units) was added to each sample and an HPLC tracewas immediately obtained followed by subsequent HPLC injections every 45min. A standard curve of the authentic C-terminal fragment was generatedto convert the peak area to percent cleavage.

Fluorogenic Peptide Studies Methods

The fluorogenic peptide NorG-E(EDANS)RPAHLRDSGK(dabcyl)GSGSG wasprepared by SPPS as described above where the EDANS was added as amodified Glu (FMOC-Glu(EDANS)-OH; AAPTec, cat. #AFE150) while dabcyl(Anaspec, cat #81800) was conjugated to the e-amino group of a lysine.This monomer was polymerized into a homopolymer with DP=20, determinedby bulk light scattering. Note that the fluorogenic peptide sequence didnot run as a polymer on the SEC column needed for SEC-MALS. Blendcopolymers with a PEG monomer were prepared by first assessing the rateof polymerization of the two monomers. At the concentration of monomerstudied, the PEG monomer was quick to polymerize (complete within 15min), while the fluorogenic substrate polymerized at a rate of 1.78monomers per hr. To ensure reasonable interdigitation of the twomonomers in the random blend copolymer, the PEG monomer was added viasyringe pump at appropriate rates to prepare peptide: PEG polymers at aratio of 1:19, 5:15, 10:10, 15:5 and 19:1 as described in Table 9.Cleavage of the homopolymer monomer and blend copolymers (40 μM) by thenoted protease in (at 25 nM) in reaction buffer (50 mM Tris (pH 7.4), 1mM ZnCl₂, 150 mM NaCl, 5 mM CaCl₂) was monitored by measurement offluorescence in a plate reader or fluorimeter. The proteases, MT1-MMP(catalytic domain; Calbiochem, cat. #476935), MMP-9 (catalytic domain;Enzo Life Sciences cat. #BML-AW360-0010), thermolysin (Promega, cat.#V4001), trypsin (Gibco Life Tech., cat. #15090-046) and Pronase (Roche,cat. #10165921001) were purchased from commercial sources.

Graphs showing the determination of concentration of fluorogenicmaterials in water at 340 nm (EDANS absorbance) and at 378 (dabcylabsorbance) were obtained. The concentrations used in proteolysisstudies are the average concentration obtained at the two wavelengths. Aspectra showing the excitation and emission for authentic products on afluorimeter were obtained. Max excitation and emission were determinedto be at 340 nm and 495 nm, respectively. A standard curve was obtainedon a fluorimeter (used for monitoring cleavage kinetics of homopolymersand all cleavage reactions by MMP-9 due to higher sensitivity than theplate reader). A plot showing a standard curve on a plate reader (usedfor assessing cleavage kinetics of monomers for enzymes other thanMMP-9) was obtained.

TABLE 9 Tabulated values for the preparation of reasonablyinterdigitated random copolymers. OEG Initiator Total Calculated Rate ofOEG:Peptide Peptide Solution Solution Reaction Reaction OEG Ratio VolumeVolume Volume Volume Time Addition Theoretical (m:n) (μL) (μL) (μL) (μL)(hr) (μL/hr) M_(n) M_(n)  0:20 368.4 0 20 388.4 11.2 — 46,200 43,000 5:15 276.3 92.1 20 388.4 8.4 10.9 36,500 36,000  10:10 184.2 184.2 20388.4 5.6 32.8 26,700 26,000 15:5 92.1 276.3 20 388.4 2.8 98.4 16,90016,300 19:1 18.4 350 20 388.4 0.56 — 8,670 8,700

Given that OEG monomer is fast to polymerize (at the concentration used,the OEG monomer polymerizes to DP=20 in less than 15′), this monomer isdoped into a stirring solution of the substrate monomer and theinitiator via a syringe pump in a glove box. For the 19:1 blendcopolymer, the OEG monomer was added in one portion to the reactionvessel after ˜2 hr. The initial concentration of the OEG monomer,substrate monomer and catalyst were 0.012 M, 0.012M and 0.6 mM,respectively. The bulk M_(n) obtained for the homopolymer was 43,000(DP=19). Given that the monomers and imitators were taken from the samepots, it is assumed that the ratios of m:n are as indicated.Nevertheless, M_(n) values obtained by batch mode SLS are listed and arein good agreement with the theoretical values calculated from theintended m:n ratios.

Evaluation of Two Additional Polymerized Peptide Substrates

To test whether the strategy could be extended to different proteasesand to peptide sequences other than the highly charged Tat and Arg8sequences, two additional peptide substrates were polymerized.Importantly, the two peptides each had a more extensive sampling ofamino acid side chains compared to the CPP sequences and were optimizedsubstrates for degradation by two different enzymes: a serine proteaseand a metalloprotease.

The generality of the approach was examined by preparing a peptidesubstrate for thrombin, a coagulation factor protease. A monomer bearingthe thrombin substrate sequence (GALVPRGS, SEQ ID NO:73) was readilyprepared via SPPS with a short, water-solubilizing peptide sequence(GERDG, SEQ ID NO:74) at the C-terminus (FIG. 12), and was polymerizedby ROMP to several degrees of polymerization (characterization data forpolymers are given in Table 4). The monomer peptide and homopolymerswere treated with thrombin, and the resulting product mixture wasanalyzed by RP-HPLC (FIG. 13). These analyses indicate that themonomeric peptide was readily degraded by thrombin, as evidenced by thedisappearance of the monomer peak and corresponding appearance ofproduct peaks, however, homopolymers at several degrees ofpolymerization were resistant to cleavage relative to the monomer,albeit not completely shut off from proteolysis, confirming thegenerality of the approach (FIGS. 7A and 7B).

A chromatogram showing RP-HPLC characterization of the purified monomerat a gradient of 15-30% buffer B was obtained. ESI-MS (electrosprayionization mass spectrometry) confirmed the identity-calculated: 1473.5m/z, obtained: 1471.6 m/z. A spectra showing polymerization of thrombinsubstrate monomer as determined by ¹H NMR was obtained. A chromatogramshowing authentic product resulting from a cleavage reaction(GSGERDG-NH₂) was obtained with gradient of 0-20% Buffer B. ESI-MSconfirms the identity-calculated: 675.7 m/z, obtained: 674.4 m/z. Astandard curve used to determine the concentration of products, whichcompares the concentration of the authentic product to the correspondingpeak area in RP-HPLC chromatograms with gradient 0-80% Buffer B, wasobtained.

An optimized peptide substrate sequence for a cancer-associatedmembrane-bound matrix metallopro-tease (MMP) was examined. Here, theN-terminal Cys residue was omitted from the optimized peptide sequence,CRPAHLRDSG (SEQ ID NO:75), because free thiols (lacking protectinggroups) are difficult to polymerize by ROMP, due to coordination to theinitiators. Since this peptide was not expected to function in anorthogonal bioactivity assay, as for the CPP studies above, the sequencewas prepared as a fluorogenic substrate, to readily and rapidly quantifycleavage events at low concentrations of material to obtain detailedkinetic information.

Kinetic and Enzymatic Assays

In kinetic assays, the fluorogenic monomer was readily cleaved by anassortment of proteases (FIGS. 7C and 7D), but not by MMP-9 for which itis not a substrate (FIG. 17). In contrast, the homopolymer (DP=20)exhibited very little cleavage upon treatment with multiple proteases,as seen in the 24-h time course plots (FIG. 7B). Initial enzymaticreaction rates (V₀), obtained by monitoring each reaction for the first40 min (less than 25% cleavage seen for all materials), indicated thatthe monomer was cleaved 17- to 95-fold faster than the homopolymer atcomparable peptide concentrations (Table 10). Michaelis-Menten plotswere obtained for the cleavage of the monomer by MT1-MMP, yielding aspecificity constant (k_(cat)/K_(m)) of 0.52±μM⁻¹ min⁻¹ and a K_(m) of11 μM. A Michaelis-Menten plot of the homopolymer time-course datarevealed that saturation kinetics had not been reached at ˜7 times thecalculated K_(m) of the monomer, as suggested by the near linear fit tothe data obtained. Note that the fluorescence observed in assays of thehomopolymer approached the lower limit of detection, resulting in largestandard errors in the data. Moreover solubility limits of thehomopolymer prevented a full Michaelis-Menten plot from being generated.Nevertheless, these data clearly indicated that the protease exhibitedlower affinity for and activity on the peptide substrate when it wasincorporated into a brush polymer.

A graph showing the time course of the fluorogenic substrate monomer asmonitored by ¹HNMR was obtained. At each time point, integration of theolefinic proton of the monomer 6 6.32 is tabulated and normalizedrelative to a time point at which the reaction is complete (finalspectrum; t=12 hr). This value is converted into a number of monomersconsumed by assuming that the integration value at t=12 represents theconsumption of 20 monomers and the value at t=0 hrs represents that ofno monomers consumed. The strategy for preparing random blend copolymersof the fluorogenic substrate is shown. A plot showing the number ofmonomers consumed as calculated in A vs. time was obtained. The slope ofthe line is the rate of monomers consumed per hour.

TABLE 10 Initial velocities (V₀) for the proteolysis of fluorogenicmonomer and homopolymer by assorted proteases Monomer V₀ Homopolymer V₀Protease (μM min⁻¹) (μM min⁻¹) MT1-MMP 0.11 ± 0.01 0.0066 ± 0.002Thermolysin 0.27 ± 0.03 0.0055 ± 0.001 Trypsin 0.18 ± 0.01  0.002 ±0.001 Pronase  0.2 ± 0.01 0.0021 ± 0.001 MMP-9  0.005 ± 0.0002 — ^(a)Theterms monomer and homopolymer correspond to the fluorogenic homopolymerand polymers. The fluorogenic peptide is optimized so that it is not asubstrate for MMP-9. No fluorescence was detected during treatment ofthe homopolymer with MMP-9.

Example 6: Proteolytic Susceptibility of Peptide-Containing BrushPolymers

The origin of proteolytic resistance was studied, and it was alsorecognized that, in certain circumstances, it might be disadvantageousto render a peptide entirely refractory to proteolysis, for example, inthe case of a peptide sensor for a protease, or when designing a devicethat targets tissue or releases a drug in response to proteolyticdigestion. Therefore, the proteolytic susceptibility of the fluorogenicsubstrate for MT1-MMP described above was evaluated. The proteolyticresistance of homopolymers may result from packing or other stabilizingpeptide-peptide interactions, leading to steric protection againstenzymatic cleavage. This picture led to the notion that proteolyticsusceptibility would be restored by spacing the peptides out along thepolymer backbone.

Spacing was accomplished by preparation of random blend copolymers thatincorporated a monomer “spacer” or “diluent” at varying blend ratios(FIG. 8A). The spacer chosen was a water-soluble OEG monomer, which isinert to proteolytic enzymes. Random blend copolymers (total DP=20) wereprepared at substrate to OEG ratios of 1:19, 5:15, 10:10, and 15:5 (FIG.8A). A general trend emerged in which proteolytic activity of MT1-MMPwas greatest when more spacers were incorporated. Indeed, the 1:19 blendpolymer proved to be as susceptible to proteolytic degradation as thesubstrate monomer (FIG. 8B). These data indicated that the protectionfrom proteolysis observed in the systems did not result simply from theattachment of the peptide to a high molecular weight polymer, but ratherfrom its arrangement into a high-density peptide brush.

Example 7: Molecular Dynamics Simulations

Computational Methods

Details of the polymer constructs that were simulated are as follows. Inall cases, the polymer backbone was composed of 10 norbornene units,flexibly linked by olefin bonds, with a 1:1 mix of cis and trans units.In the homopolymer, each norbornene residue was linked to the N-terminusof the fluorogenic peptide NorGlyE(EDANS)RPAHLRDSGK(DABCYL)GSGSG) (FIG.9A), where the EDANS and DABCYL fluorophores are linked to E and Kresidues, respectively. The C-terminus of each peptide was amide capped.In the 5:5 blend copolymer, five of these peptide-dye chains were linkedto norbornenes 1, 2, 5, 6, and 10 (counting from the end of the phenylring); and five OEG chains, each with four ethylene glycol units, werelinked to the remaining norbornenes (FIGS. 9G and 9H). In the 9:1 blendcopolymers, all positions are occupied by OEGs except that the tenth orfifth norbornene (FIGS. 9C and 9D or 9E and 9F) is occupied by thefluorogenic peptide.

All-atom molecular dynamics simulations were performed to study theconformations of the simulated polymers, using the explicit water modelTIP3P91 and the Gromacs 4.6 software package. All bonded andLennard-Jones terms of the polymer backbone and dye moieties wereassigned by the General Amber Force Field (GAFF) and partial atomiccharges were assigned using AM1-BCC. Parameters from the Amberff99SB-ILDN force field were assigned to the peptide components. Allsimulations started from extended polymer backbone and peptideconfiguration and were performed using periodic boundary conditions.Each polymer construct was solvated in a cubic simulation box with edgelengths set to the longest dimension of the molecule plus 2 nm. This ledto box sizes with edge lengths of 10-15 nm. The systems were firstenergy minimized with the steepest-descent algorithm, and thenequilibrated for 10 ns under constant volume and temperature conditionsand then another 10 ns under constant temperature and pressureconditions. The Particle-Mesh-Ewald (PME) method was used forelectrostatic interactions, and the cutoff distance of the Lennard-Jones(LJ) interactions was 10 Å. In some simulations, a heat-cool cycle wasused immediately after the equilibration phase to boost the systems outof local energy minima and search for additional stable conformationalstates. Here, the temperature was increased from 300 to 500 K linearlyover 2 ns; the simulation was run for 1 ns at 500 K; and the temperaturewas then reduced back to 300 K linearly over 2 ns, and kept at 300 K for95 ns of production dynamics. For comparison, regular MD simulations atconstant 300 K were also performed for 100 ns following the sameequilibration phase. The two 9:1 blend copolymers without the dyecomponents were stimulated, to verify that the dye molecules do notinfluence the major conclusions. For these simulated polymers, one Cl⁻-counterion was added in order to neutralize the +1 charge.

Molecular Dynamic Simulation Methods

Here, MW is molecular weight (Da); and N, the number of constituentmonomers, is computed as the number of norbornene monomers (10) plus thenumber of amino acid residues (number of peptides×17) plus the number ofethylene oxide units (number of OEG chains×4) plus the number of dyemoieties (either 2 or 0). Each simulation began with the molecule in anartificial extended conformation, with a straight norbornene chain andwith straight peptides and OEGs arrayed at right angles to thenorbornene chain. After an initial 20 ns equilibration phase at 300K,each simulation was split and continued in two ways. One simulation wassimply continued for another 100 ns at 300K. The other was randomizedfurther by briefly heating it to 500K, cooling it back to 300K, and thencontinuing it at 300 K for the rest of the 100 ns production simulation.

In every simulation, the initial extended structure collapsed quicklyinto a much more compact conformational ensemble. The collapse isevident from an rapid early drop in the radii of gyration, Rg. Thevalues of Rg then changed little for the runs that continued at 300K(blue and green). For simulated polymers 1 and 2, which contain ten andfive peptides, respectively, the heatcool cycle (red and orange)generated a spike in Rg, followed by a further reduction in Rg below thevalue seen for the standard 300K runs. Thus, for these two molecules,the heat-cool cycle allows the polymer construct to anneal into an evenmore compact conformation. For simulated polymers 3-6, which have onepeptide and nine PEGs, the heat-cool cycle changed Rg but did notconsistently lead to a lower value. Thus, the heat-cool cycle did notseem to lead to greater compaction of these one-peptide constructs.

Radii of Gyration (Rg) Methods

The values of Rg observed here may be put into context by comparing themwith the values associated with folded proteins having a similar numberof residues or molecular weight. Thus, simulated polymer 1, with 200monomers and molecular weight of 23.2 kDa, has final Rg values of1.8-2.0 nm. For comparison, folded proteins with 151-200 residues haveRg values ranging approximately from 1.6-1.8 nm, and the proteinsribonuclease and chymotrypsinogen alpha, with molecular weights of 17and 38 kDa, have Rg values of 1.5 and 1.8 nm, respectively. Althoughsimulated polymers 3-6, have molecular weights about four times smaller(5-6 kDa), their Rg values of ˜1.2 nm are not even two-fold smaller,suggesting that these single-peptide constructs do not compact astightly as the more peptide-rich 1 and 2.

Conformational fluctuations of the simulated molecules were quantifiedby using overall rotations and translations to optimize the overlay ofeach MD snapshot on the final snapshot (120 ns) of the corresponding MDtrajectory, and computing the root-mean-square deviations (RMSDs) of theoverlaid snapshots relative to the final snapshots, as a function oftime. The RMSD values in all cases remained less than 1.5 nm in thefinal 100 ns of each simulation, and in many cases, they remained below1.0 or even 0.4 nm, particularly for the trajectories which include theheat-cool cycle. Presumably, the heat-cool cycle in these cases allowedthe structures to find a particularly stable conformational state. Thestructural fluctuations observed here are larger than those typicallyobserved in similar MD simulations of stably folded, naturally occurringproteins, but the lower end of the present range is commensurate. Itshould be noted that the final conformations generated from the simple300K simulations and from the corresponding heat-cool simulations arequite different from each other: for simulated polymers 1 and 2, theRMSDs between these conformer are 1.35 and 1.32 nm respectively.

TABLE 11 hydrogen bond counts of simulated polymers 1-6, averaged overthe last 40 ns of the heat-cool trajectories. Polymer 1 Polymer 2Polymer 3 Polymer 4 Polymer 5 Polymer 6 AA-AA 88 33 7 6 8 5 EDANS-AA 249 0 1 — — DABCYL-AA 7 3 0 0 — — Peptide-peptide 119 45 7 7 8 5 PEG-AA 02 1 1 2 0 NOR-AA 2 1 2 0 0 2 EDANS-OEG 0 0 0 0 0 0 DABCYL-OEG 0 0 0 0 00 EDNAS-NOR 1 1 0 0 0 0 DABCYL-NOR 0 0 0 0 0 0 Peptide-nonpeptide 3 4 31 2 2

Representative conformations of simulated polymers 1-4 were obtained byapplying an RMSD-based clustering algorithm to the last 40 ns of therespective heat-cool simulations. Homopolymer 1 and the 5:5 copolymer 2collapsed into an elongated globule, with their peptide chains tangledaround the polymer backbone. The single peptide chains in the 1:9copolymers, 3 and 4, are visible as relatively isolated components atthe surface of these peptide-polymer constructs. Salt-bridges andoccasional segments of alpha-helix were observed (FIGS. 42A-42D).

Molecular Dynamic Simulations with the Random Blend Copolymers

To further examine the proteolytic susceptibility trends observed withthe random blend copolymers of the fluorogenic substrate and OEG moiety,a series of molecular dynamics simulations were performed, examininganalogous blend copolymers with discrete structures and no dispersity(i.e., a single molecular entity was modeled for each structuralanalogue). For computational simplicity, all polymers were constructedin silico to have a DP of 10, instead of 20, in four key arrangementsmeant to best simulate idealized scenarios: a homopolymer of tenrepeated fluorogenic substrates (FIGS. 9A and 9B); two blend copolymerswith an OEG:peptide ratio of 9:1, one having the peptide at one end ofthe polymer (FIGS. 9C and 9D), and the other having the peptide atposition five (FIGS. 9E and 9F); and one with an intermediatepeptide:OEG ratio of 5:5 (FIGS. 9G and 9H).

Simulations were performed on each structure, starting with the moleculein an artificial, extended conformation with a straight norbornylbackbone, extended peptides, and OEG brushes arrayed at right angles tothe polymer backbone. In the simulations, each structure wasequilibrated for an initial 20 ns at 300 K, after which each simulationwas split and continued in two ways: one simulation for each moleculewas continued for 100 ns at 300 K and the other was further randomizedby a single heating (500 K) and cooling (300 K) cycle before continuingat 300 K for remainder of the 100 ns simulation.

In every simulation, the initial extended structure of each moleculecollapsed quickly into a more compact conformational ensemble.Representative conformations of each construct were obtained by applyinga root-mean-square deviation (RMSD)-based clustering algorithm to thelast 40 ns of the respective heat-cool simulations. In these structures,the homopolymer and 5:5 copolymer collapsed into an elongated globule,with their peptide chains tangled around the polymer backbone. Incontrast, the single peptide chain in the 9:1 copolymers, were visibleas relatively isolated components at the surface of the constructs. Adetailed analysis of differences in radius of gyration of each structureand also on conformational fluctuations in the structures as quantifiedby RMSDs was determined. A graph showing radii of gyration (Rg) ofsimulated polymer 1 under constant temperature condition (300K) andusing simulated annealing procedure was constructed. A graph showing Rgof simulated polymer 2 under constant temperature condition (300K) andusing simulated annealing procedure was constructed. A graph showing Rgof simulated polymer 3 under constant temperature condition (300K) andusing simulated annealing procedure was constructed. A graph showing Rgof simulated polymer 4 under constant temperature condition (300K) andusing simulated annealing procedure was constructed. A graph showing Rgof simulated polymer 5 under constant temperature condition (300K) andusing simulated annealing procedure was constructed. A graph showing Rgof simulated polymer 6 under constant temperature condition (300K) andusing simulated annealing procedure was constructed. A graph of RMSD(root-mean square deviation) of simulated polymer 1 was obtained. RMSDvalues were calculated with respect to the structure in the last framein each run. The program g_rms in Gromacs3 was used to generate therotational and translational overlays and compute RMSD values.Similarly, a graph of RMSD of simulated polymer 2 was constructed. agraph of RMSD of simulated polymer 3 was constructed. Similarly,k agraph of RMSD of simulated polymer 4 was constructed. Similarly, a graphof RMSD of simulated polymer 5 was constructed. Similarly, a graph ofRMSD of simulated polymer 6 was constructed. A representativeconformation of simulated polymer 1 was generated. Similarly, arepresentative conformation of simulated polymer 2 was constructed. Arepresentative conformation of simulated polymer 3 was constructed. Arepresentative conformation of simulated polymer 4 was constructed.

Role of Hydrogen Bonding to Facilitate Compression or Tangling of thePeptide-Containing Structures

The role of hydrogen bonding in facilitating the compression or tanglingof the peptide-containing structures by computing the numbers ofintrasolute hydrogen bonds during the last 40 ns of each heat-cooltrajectory (Table 11) was examined. The homopolymer averaged 0.5 aminoacid-amino acid peptide bonds per residue (88 interactions over 17residues per monomer), which was about half the ratio typically of afolded protein. The three copolymers averaged fewer (0.4) hydrogen bondsper residue, with very few hydrogen bonds to the OEG moieties (less thanfour in all cases). Overall, these hydrogen bond counts were consistentview that although the polymers collapsed, they were not as wellstructured as typical globular proteins and the inclusion of OEG unitslead to a decrease in hydrogen bonding. Thus, the addition of OEG unitseffectively “diluted” the density of the peptide-brush by reducing theoverall degree of hydrogen bonding in the polymer structure. Moreover,the OEG moieties blocked what would otherwise be stabilizinginteractions among the peptides brushes without compensating with newOEG-peptide interactions.

Accessibility of Peptide Components to Large and Small Molecules

The accessibility of the peptide components of the various constructs tolarge and small molecules was examined by computing their time-averagedprobe-accessible surface area (SA), using a large (3.14 nm) probe spherewhose size is similar to that of a protein, and a small (0.14 nm)water-sized probe sphere (FIG. 9I). A general trend was seen with thelarge probe, where larger surface accessibility was seen as the peptidecontent decreased. The greatest accessibility was observed for the 9:1copolymer with the peptide at position 5. This was the closestrepresentation to the experimental 19:1 random blend copolymer (based onthe polymerization method, Table 9), which was nearly as susceptible toproteolytic degradation as the peptide monomer in vitro. In contrast,the probe-accessible surface areas obtained with the water-sized probewere relatively uniform across all constructs. These results indicatedthat the peptides in all five constructs maintained similaraccessibility to small molecules, like water, but that the tighterpacking of the more peptide-rich constructs reduced accessibility toprotein-size molecules, such as the proteases examined experimentally.Moreover, these data indicate that peptides whose function depends oninteraction with a small molecule or a receptor with a relativelyaccessible binding site exhibited ample binding to the peptidesubstrates in any of the constructs considered, including thehomopolymer. The implication is that the bioactivity of many peptideswill be maintained after polymerization of the sequence into ahigh-density brush. For peptides whose function depends on interactionwith proteins or macromolecules with tight or cramped binding pockets(such as a protease), polymerization into a high-density brush polymermay impede function, as is consistent with the data presented herein.

The simulation results indicated that all of the constructs studiedcollapsed into fairly compact globular conformations, and that a higherpeptide content lead to formation of more stabilizing intramolecularhydrogen bonds and reduced accessibility of the peptides to proteins insolution. This picture was qualitatively consistent with theexperimental observation that constructs with high peptide content arebetter protected from enzymatic degradation. Although the simulationsare subject to error due to their limited duration and uncertainties inthe force field, they yield a detailed representation of the systemsunder study and offer a plausible explanation for the key experimentalresults.

Example 8: Activating Peptides for Cellular Uptake Via Polymerizationinto High Density Brushes

Materials

All amino acids used to prepare peptides by solid phase peptidesynthesis (SPPS) were obtained from AAPPTec and NovaBiochem. Unlessotherwise noted, all other compounds and materials were purchased fromSigma Aldrich and used without further purification. The GSGSGmonomer,[1] GSGSG peptide control, Tat peptide control, and OEG monomerwere synthesized. The polymerization initiator, SI,(H2IMES)(pyr)2(Cl)2Ru═CHPh was also prepared by a published protocol.[3]Analytical scale reverse-phase HPLC (RP-HPLC) was performed on a JupiterProteo90A Phenomenex column (150×4.60 mm) equipped with a Hitachi-EliteLaChrom L2130 pump and a UV-Vis detector (Hitachi-Elite LaChrome L-2420)monitoring at 214 nm. Peptides were purified on a preparative-scaleJupiter Proteo90A Phenomenex column (2050×25.0 mm) using an Armen SpotPrep II System. In all cases, peptides were purified and analyzed forpurity using a gradient buffer system in which Buffer A is 0.1% TFA inwater and Buffer B is 0.1% TFA in acetonitrile. Polymer dispersities(Mw/Mn) and molecular weights (Mn) were determined by size-exclusionchromatography coupled with multiangle light scattering (SEC-MALS). Tothis end, SEC was performed on a Phenomenex Phenogel 5 u 10, 1K-75K,300×7.80 mm column in series with a Phenomenex Phenogel 5 u 10,10K-1000K, 300×7.80 mm column, which ran with 0.05 M LiBr in DMF as therunning buffer (flow rate of 0.75 mL/min) using a Shimadzu pump. Theinstrument was also equipped with a MALS detector (DAWN-HELIO, WyattTechnology) and a refractive index (RI) detector (Wyatt Optilab T-rEXdetector). The entire SEC-MALS set-up was normalized to a 30K MWpolystyrene standard. All concentrations of fluorescein-labeledmaterials were obtained by measuring UV absorbance of the fluorophore(at 495 nm) on a ThermoScientific Nanodrop 2000c and fitting the dataobtained to the standard curves. The emission profiles of all materialswere equivalent at these concentrations (ex: 495 nm, em: 520 nm).Fluorescent data was recorded on a Perkin Elmer EnSpire Multimode PlateReader. A Varian Mercury Plus spectrometer was used to obtain all 1H(400 MHz) NMR spectra. Chemical shifts are reported in ppm relative tothe DMF-d7 residual proton peaks. Flow cytometry was performed on anAccuri C6 flow cytometer set to default “3 blue 1 red” configurationwith standard optics and slow fluidics (14 μL/min).

Polymerizations Methods

All polymerizations were carried out in a glove box under N₂ (g). Atypical protocol (FIG. 23) used to generate a polymer with DP (or “m” inFIG. 19A)=8 involved mixing the monomer (0.0125 mmol, 8 equiv., 25 mM)with the catalyst initiator (FIG. 1A) (0.00156 mmol, 1 equiv., 3.1 mM)in dry DMF (0.5 mL). For each peptide monomer whose polymerization hasnot been reported previously in the literature, the time course of thepolymerization in DMF-d7 by ¹H NMR was followed to confirm completeconsumption of the monomer and to determine the time period required toreach completion (FIG. 24). To track cellular uptake, each polymer wasend-labeled with a copy of fluorescein by treatment with a chaintransfer agent (1.5 equiv.) for 2 hrs, followed by termination withethyl vinyl ether (10 equiv.) for 1 hr at room temperature. Blockcopolymers used in the GSGSG series were prepared by first polymerizingthe peptide monomer to completion prior to adding and polymerizing theOEG monomer. The block copolymers were prepared in this manner to obtainan accurate estimation of the DP or m of the functional peptide block(using a dn/dc of 0.179 in DMF) since the peptide and OEG blocks havedifferent dn/dc values in DMF. The accessory, water-solubilizing OEGblock (dn/dc=0.11 in DMF) was then polymerized second and ratios of thedn/dc values of the two blocks were used to calculate values for thisblock (i.e., the dn/dc for m=8, 15, 30 and 60 was 0.131, 0.141, 0.151and 0.161 based on the ratio of peptide:OEG used). Following completionof the second block, the resulting polymers were end-labeled with thefluorescein chain transfer agent and the active catalyst was thenterminated with ethyl vinyl ether as described above.Fluorescein-labeled polymers were treated with NH₄OH (aq) for 30 min toremove the pivolate protecting group. The resulting polymers were thendirectly characterized by SEC-MALS to obtain the polymer molecularweight (M_(n)), dispersity and degree of polymerization (DP).

All polymers were then precipitated with cold ether and collected bycentrifugation. For the side-chain protected peptide monomers, theresulting powder was dissolved in 2 mL of a mixture of TFA/H₂O/TIPS(95:2.5:2.5) and stirred for 4 hours at room temperature. The productwas precipitated with cold ether, collected by centrifugation and dried.In preparation for in vitro studies, all polymers were washed (3×) withcold ether (to remove the Ru catalyst) and then dissolved in DPBS anddialyzed in dialysis cups with a molecular weight cut-off value of 3500(Thermo Scientific, cat. #69552) in an effort to remove any residualmonomer or catalyst.

Cell Culture Methods

HeLa cells were purchased from ATCC (CCL-2). Cells were cultured at 37°C. under 5% CO2 in Dulbecco's Modified Eagle Medium containing phenolred (DMEM; Gibco Life Tech., cat #11960-044) that was supplemented with10% fetal bovine serum (Omega Scientific, cat # FB02) and with 1×concentrations of non-essential amino acids (Gibco Life Tech., cat#11140-050) sodium pyruvate (Gibco Life Tech., cat. #11360-070),L-Glutamine (Gibco Life Tech., cat. #35050-061), and the antibioticspenicillin/streptomycin (Corning Cellgro, cat. #30-002-C1). Cells weregrown in T75 culture flasks and subcultured at ˜75-80% confluency (every˜3-4 days).

Flow Cytometry Methods

HeLa cells were plated at a density of 90,000 cells per well of a24-well plate 18 hrs prior to treatment with the material of interest.Materials dissolved in Dulbecco's Phosphate Buffered Saline (DPBSwithout Ca2+ or Mg2+; Corning Cellgro, cat. #21-031-CM) at 10× thedesired concentration (where concentration is with respect tofluorophore concentration to ensure propare comparison of each moleculartransporter) were added to the wells and the plates were incubated for30 min at 37° C. The medium was then removed and the cells were washed2× with DPBS and then incubated 3× for five minutes each with heparin(0.5 mg/mL in DPBS; Affymetrix, cat. #16920) to remove anymembrane-bound, non-internalized material, and finally rinsed again withDPBS. The cells were then trypsinized (0.25% trypsin in DPBS; Gibco LifeTech., cat. #15090-046) for 10 min, cold medium was added, and the cellswere transferred to Eppendorfs. The suspended cells were centrifuged topellets and then resuspended in a minimal amount of cold DPBS. Flowcytometry data (10,000 events on three separate cultures) was thenacquired.

Live-Cell Confocal Microscopy Methods

HeLa cells were plated on glass-bottom 24-well plates at a cell densityof 90,000 cells per well 18 hrs prior to treatment with the compound ofinterest. The medium was removed and then replaced with medium lackingphenol red (Gibco Life Tech., cat#31053-028) to minimize backgroundfluorescence. Materials dissolved in DPBS (at 10× the desiredconcentration, 2.5 μM, where concentration is with respect tofluorophore) were added to the wells and the plates were incubated for30 min at 37° C. The medium was then removed and the cells were rinsed2× with DPBS, and then fresh medium (phenol red-free) was added to eachwell. Live cells were imaged on an Olympus FV1000 confocal microscopewith a chamber at 37° C. under 5% CO2 via a Z-stack analysis (set to 1μm slices) using identical instrument settings for each image and a 40×objective.

Cell Viability Assay Methods

The cytotoxicity of materials was assessed using the CellTiter-Blue®assay (Promega, cat # G8081), which measures the metabolic reduction ofresazurin to resorufin via fluorescence. For these studies, HeLa cellswere plated at a density of 3,500 cells per well of a 96-well plate 18hrs prior to treatment. Materials dissolved in DPBS at 10× the desiredconcentrations were added to the wells along with a 10% DMSO positivecontrol. Cells were incubated for 72 hrs at 37° C. Note thatconcentration for all toxicity measurements is with respect to peptideconcentration to ensure that all peptides and polymers are fairlycompared with respect to their therapeutic components. There was also nofluorophore present on the polymers or peptides used in theseexperiments to minimize potential background signals in the fluorescentmeasurements. The medium was removed and 80 μL of fresh medium lackingphenol red was added. To this was added 20 μL of the CellTiter-Blue®reagent and the cells were then incubated for 2 hrs prior to measuringfluorescence using 560 nm excitation and 590 nm emission. Thefluorescence measurements were corrected for background fluorescencefrom the CellTiter-Blue® reagent by subtracting the fluorescence readingof wells treated with the reagent in the absence of cells. Fluorescencevalues were then referenced as a percentage of the value obtained forthe DPBS vehicle control.

Circular Dichroism Methods

UV-Vis circular dichroism (CD) was used to evaluate whether thesecondary structure of KLA, a mixture of random coil and alpha helix, ismaintained upon polymerization. The peptide and polymer were dissolvedin DPBS to a final concentration of 100 μM (with respect to peptideconcentration). CD spectra were measured using an Aviv 215 spectrometerand each sample was measured from 190 to 260 nm with a slit width of 1nm, scanning at 1 nm intervals with a is integration time. Measurementswere taken 3× at 25° C. and then averaged to give the spectra in FIGS.30A-30D.

JC-1 Mitochondrial Integrity Assay

The mitochondrial membrane potential was measured using the MitoProbe®JC-1 assay kit (Life Technologies, cat #M34152), which measures themembrane potential using the JC-1 dye. The JC-1 dye forms J aggregatesin healthy mitochondria that are red fluorescent, but cannot form the Jaggregates when the mitochondria are disrupted, leading to a decrease ofred fluorescence and an increase of green fluorescent J monomers in thecytosol. HeLa cells were plated at a density of 90,000 cells per well ofa 24-well plate 18 hrs prior to treatment. Materials dissolved in DPBSat 10× the desired concentrations (where concentration is with respectto peptide and no fluorophore is present) were added to the wells andincubated for 30 minutes. The materials were removed and the cells werewashed with DPBS, then medium was added to the cells followed byincubation with 10 μL of a 200 μM solution of JC-1 dye to give a finalconcentration of 2 μM. To one set of wells, 2 μL of 50 mM carbonylcyanide 3-chlorophenylhydrazone (CCCP) was added to give a finalconcentration of 50 μM. The small molecule CCCP is used as a positivecontrol because it can associate with and depolarize mitochondrialmembranes. The cells were incubated for 30 more minutes. The medium wasthen removed and the cells were washed 2× with DPBS and then incubated3× for five minutes each with heparin (0.5 mg/mL in DPBS; Affymetrix,cat. #16920), and finally rinsed again with DPBS. The cells were thentrypsinized (0.25% trypsin in DPBS; Gibco Life Tech., cat. #15090-046)for 10 min, cold medium was added, and the cells were transferred toEppendorfs. The suspended cells were then centrifuged to pellets andthen resuspended in a minimal amount of cold DPBS. Flow cytometry data(10,000 events on three separate cultures) was then acquired.

Apoptosis Assay Methods

The expression of caspase enzymes, markers of apoptotic cells, in thecells treated with KLA materials was assessed using the Apo-One® assay(Promega, cat # G7790), which measures the expression of caspase 3/7, amarker of apoptosis, using a fluorogenic substrate for those enzymes.HeLa cells were plated at a density of 5,000 cells per well of a 96-wellplate 18 hrs prior to treatment. Materials dissolved in DPBS at 10× thedesired concentration (10 μM, where concentration is with respect topeptide and no fluorophore is present) were added to the wells. Cellswere incubated for 30 minutes at 37° C. The medium was removed the cellswere washed 3× with DPBS. 100 μL of the Apo-One® reagent was added tothe cells and to wells that contain no cells to get a baseline reading.The plate was mixed for 30 seconds using a shaker followed by incubationat 37° C. After 3 hrs of incubation, the fluorescence was measured usingan excitation of 499 nm and emission at 521 nm. The baselinefluorescence was subtracted from the fluorescence values for the wells,and fluorescence values were then referenced as a percentage of thevalue obtained for the DPBS vehicle control.

Mechanistic Studies by Flow Cytometry

For mechanistic studies, cells were plated and treated as described inthe previous flow cytometry experiments, except here cells werepreincubated with the indicated compound for 30 minutes at 37° C. priorto addition of the cell-penetrating material. The followingconcentrations were used: 80 μM dynasore (Enzo Life Sciences, cat.#270-502-M005) and 9.5 mM MβCD (Fischer Scientific, cat # AC377110050).For studies at reduced temperature, cells were incubated at 4° C. for 30min prior to and during incubation with the compound of interest. Forthe reduced temperature studies only, all subsequent washes andmanipulations were also done with ice-cooled media and other materials.Data is reported as the normalized mean fluorescence, which is the meanfluorescence yielded by the material divided by the mean fluorescencefrom the vehicle control. Each measurement was preformed 3× on at leastthree separate subcultures.

RP-HPLC Analysis of Proteolytic Susceptibility

The percent of intact GSGSGKK (SEQ ID NO:17), GSGSGRR (SEQ ID NO:15) orKLA peptides and polymers after incubation with trypsin (Gibco LifeTech., cat. #15090-046) or Pronase (Roche, cat. #10165921001) wasassessed by comparing RP-HPLC chromatograms. In these experiments, eachpeptide or polymer (50 μM, where concentration is with respect topeptide content to ensure fair comparison between the peptides andpolymers) was incubated with each protease (at 1 μM) for 3 hrs. At thispoint, the proteases were heat denatured at 65° C. for 15 m and theresulting solution was immediately injected onto an analytical RP-HPLC.Given that treatment with each protease often yielded multiple peptidefragments, a standard curve for each starting material (rather thanevery potential product) was prepared to assess the percentage of intactmaterial remaining after proteolytic digestion. Note that the standardcurves for polymers will be biased because the polymer backbone, OEGcoblock and fluorophore should remain intact after cleavage, and willtherefore comprise part of the measured peak area. Nevertheless, no newpeaks were seen in the chromatograms of any polymer post enzymetreatment, suggesting that these materials are not susceptible tocleavage by the proteases. Major fragments of the peptide controls werealso identified by ESI MS. Consistent with the notion that the polymersare protected from proteolysis, no discernable peptide fragments wereidentified by MS in the polymer reaction mixtures.

Obstacles in Peptide-Based Therapeutic and Diagnostic Agents

The chemical diversity inherent to natural and unnatural amino acidsenables the formulation of peptides that are selectively and preciselycoded for interaction with target receptors and other biologicalsurfaces. This ability has fostered the development and identificationof unique natural, semi-synthetic and synthetic peptide sequencescapable of diverse medicinal and diagnostic applications. Despite theirpromise, the clinical efficacy of many peptide-based therapeutic anddiagnostic agents is severely hampered by three key obstacles: errantproteolysis, inefficiencies in cellular uptake, and size-dependent renalclearance. A strategy for protecting peptides from proteolysis in whichpeptides are packaged as high density brush polymers via graft-throughring opening metathesis polymerization (ROMP) of peptide-based monomers,generating structures that are resistant to proteolytic degradation wasreported. This strategy does not require chemical modification of theprimary amino acid sequence and is, therefore, a facile approach toaccess formulations of protease-resistant peptides that maintain theirinherent function. Here it is demonstrated that when polymerized into ahigh density brush polymer, peptides bearing at least one Arg or Lys canefficiently penetrate cells.

The biological target of most therapeutic agents resides in the cytosolor nuclei of cells. Therefore, potential therapeutic peptides thatcannot gain entry into the interior of a cell are generally ineffective.Conventional strategies for conferring cellular uptake typically involveappending the peptide of interest to a cell penetrating peptide (CPP).CPPs, such as Tat and Arg8, are most often highly charged sequences thatcontain multiple copies of arginine (Arg). CPPs of this type have beenshown to deliver a wide variety of conjugated cargo into cells. However,materials linked to CPPs in a linear arrangement maintain theirsusceptibility to proteolytic digestion. Thus, the development ofgeneral strategies that provide the needed dual function of protectingpeptides from proteolysis while facilitating cellular entry have thepotential to change the way peptides are prepared and delivered.

Non-CPP Based Molecular Transporters

There are a number of non-CPP based molecular transporters capable oftraversing cellular membranes with cargo in tow. These constructs aremostly comprised of a nanomaterial scaffold, such as a dendrimer, whosesurface is decorated with several copies of guanidinium, the chemicalmoiety present on Arg side chains that endows CPPs with their cellpenetrating properties. Near to the goal of cell penetration by peptidepolymers, is a strategy developed by Kiessling and co-workers in whichguanidinium units are appended via a graft-to approach to a preformedpolymer prepared by ROMP. This system and close derivatives designed byTew remain the only examples of membrane penetrating polynorbornylpolymers.

The strategy reported herein is inspired by these designs, but seeks asimpler, generalizable approach specific to peptide uptake.Incorporation of a single Arg residue into the amino acid sequence of anon-CPP, and subsequent polymerization of that peptide into a highdensity brush polymer, would enable cellular uptake of these materials.This strategy may provide a new route to the development ofpeptide-based therapeutics solving two major issues; 1) degradation byproteases and 2) inefficient cellular uptake; each of which haveseverely limited (if not negated) the success of many promisingpeptide-based drug candidates. Moreover, the strategy offers keyadvantages over traditional methods for conferring cellular uptakebecause the brush polymers produced have a much higher density (weightpercentage) of the therapeutic agent and require few synthetic orpurification steps.

To test the strategy, a peptide sequence, GSGSG (SEQ ID NO: 1) wassynthesized, that does not penetrate cells and appended one or two Argresidues to the N or C terminus, reasoning that these locations wouldyield the highest likelihood of maintaining the inherent bioactivity ofan otherwise intact peptide sequence (FIGS. 19A-19B, FIG. 22 and Tables10 and 11). These peptides were prepared as fluorescein-labeled peptidecontrols and also as fluorescein-terminated brush polymers via thegraft-through ROMP strategy (FIG. 19A). To ensure solubility, polymerswere prepared as block copolymers with a second block containing an OEG(oligoethylene glycol) unit (degree of polymerization (DP) approx. 20),which does not penetrate cells alone (for polymer synthesis andcharacterization data, FIGS. 23-24 and Table 12). The relative extent ofuptake of each material in HeLa cells was quantified by flow cytometry,where concentration in these studies is with respect to fluorophore (2.5μM) to ensure direct comparison of each material's ability to transportitself and its cargo (fluorescein). In all cases, the monomeric peptidecontrols showed fluorescence signals that were indistinguishable fromthat of the vehicle control. However, peptides containing at least oneArg that were polymerized with a DP (or “m” in FIG. 1A) of approximately60, penetrated cells as efficiently as a canonical CPP (Tat). Imagesfrom live-cell confocal microscopy supported this data, in whichfluorscence signatures are observed across consecutive 1 μM Z-slices foronly polymers containing cationic residues, indicating that thesematerials are internalized and not simply bound to the surface of thecell membrane. Live-cell confocal microscopy images were obtainedshowing the average intensities from six consecutive 1 um slices of HeLacells treated with peptides and polymers (m ˜60). In each study, theconcentration of materials is 2.5 μM with respect to the fluorophore. Agraph showing flow cytometry data for the vehicle control (DPBS) wasobtained. Healthy populations (10,000 events each) were gatedidentically and referenced to vehicle (DPBS). Each measurement wasrepeated 3× on three separate cultures. The concentration for eachmaterial is 2.5 μM, where concentration is with respect to thefluorophore. A graph showing flow cytometry data for the GSGSGRR polymer(m˜8) was obtained. Healthy populations (10,000 events each) were gatedidentically and referenced to vehicle (DPBS). Each measurement wasrepeated 3× on three separate cultures. The concentration for eachmaterial is 2.5 μM, where concentration is with respect to thefluorophore. A graph showing flow cytometry data for the GSGSGRR polymer(m˜15) was obtained. Healthy populations (10,000 events each) were gatedidentically and referenced to vehicle (DPBS). Each measurement wasrepeated 3× on three separate cultures. The concentration for eachmaterial is 2.5 μM, where concentration is with respect to thefluorophore. A graph showing flow cytometry data for the GSGSGRR polymer(m˜30) was obtained. Healthy populations (10,000 events each) were gatedidentically and referenced to vehicle (DPBS). Each measurement wasrepeated 3× on three separate cultures. The concentration for eachmaterial is 2.5 μM, where concentration is with respect to thefluorophore. A graph showing flow cytometry data for the GSGSGRR polymer(m˜60) was obtained. Healthy populations (10,000 events each) were gatedidentically and referenced to vehicle (DPBS). Each measurement wasrepeated 3× on three separate cultures. The concentration for eachmaterial is 2.5 μM, where concentration is with respect to thefluorophore. A graph showing flow cytometry data for the GSGSGRR polymer(m˜8-60) was obtained.

TABLE 12 GSGSG-derived block copolymers and KLA homopolymers First Block(Peptide Second Block (OEG Block) Block) Polymer Name in M_(w)/ DP^(e)M_(w)/ Text^(a) Polymer IUPAC^(b) M_(n) ^(c) M_(n) ^(d) (“m”) M_(n) ^(c)M_(n) ^(d) DP^(e) GSGSG, m ~8 GSGSG₁₁-b-OEG₁₈ 6,560 1.012 11 (8)  12,8001.12 18 (20) GSGSG, m ~15 GSGSG₁₆-b-OEG₂₁ 9,800 1.108 16 (15) 17,0001.054 21 (20) GSGSG, m ~30 GSGSG₂₈-b-OEG₁₆ 17,700 1.125 28 (30) 23,5001.075 16 (20) GSGSG, m ~60 GSGSG₅₆-b-OEG₂₈ 35,000 1.274 56 (60) 45,1001.052 28 (20) R control, m ~8 R₁₂-b-OEG₁₅ 7700 1.17 12 (8)  13,000 1.10115 (20) R control, m ~15 R₁₈-b-OEG₂₇ 11,700 1.009 19 (15) 21,500 1.06127 (20) R control, m ~30 R₂₆-b-OEG₂₀ 16,800 1.027 27 (30) 23,700 1.04820 (20) R control, m ~60 R₅₂-b-OEG₂₀ 33,900 1.036 54 (60) 40,800 1.04620 (20) RGSGSG, m ~8 RGSGSG₁₀-b- 11,600 1.005 10 (8)  19,300 1.015 21OEG₂₁ (20) RGSGSG, m ~15 RGSGSG₁₅-b- 16,500 1.09 15 (15) 22,230 1.06 16OEG₁₆ (20) RGSGSG, m ~30 RGSGSG₂₉-b- 33,100 1.007 29 (30) 42,800 1.01427 OEG₂₇ (20) RGSGSG, m ~60 RGSGSG₆₈-b- 78,000 1.143 68 (60) 90,3001.142 26 OEG₂₆ (20) GSGSGR, m ~8 GSGSGR₁₁-b- 12,130 1.08 11 (8)  20,0001.034 22 OEG₂₂ (20) GSGSGR, m ~15 GSGSGR₁₈-b- 20,000 1.034 18 (15)26,300 1.085 18 OEG₁₈ (20) GSGSGR, m ~30 GSGSGR₂₆-b- 30,100 1.053 26(30) 35,000 1.025 14 OEG₁₄ (20) GSGSGR, m ~60 GSGSGR₇₁-b- 81,100 1.15171 (60) 90,300 1.142 26 OEG₂₆ (20) RRGSGSG, m ~8 RRGSGSG₉-b- 14,5001.018 9 (8) 20,000 1.018 16 OEG₁₆ (20) RRGSGSG, m RRGSGSG₁₅-b- 23,8001.018 15 (15) 33,700 1.019 28 ~15 OEG₂₈ (20) RRGSGSG, m RRGSGSG₃₁-b-48,500 1.006 31 (30) 53,400 1.007 14 ~30 OEG₁₄ (20) RRGSGSG, mRRGSGSG₅₂-b- 80,700 1.001 52 (60) 90,800 1.003 29 ~60 OEG₂₉ (20)GSGSGRR, m ~8 GSGSGRR₈-b- 12,500 1.052 8 (8) 19,200 1.014 19 OEG₁₉ (20)GSGSGRR, m GSGSGRR₁₈-b- 28,500 1.056 18 (15) 36700 1.078 23 ~15 OEG₂₃(20) GSGSGRR, m GSGSGRR₃₀-b- 47,000 1.27 30 (30) 48,200 1.041 19 ~30OEG₁₉ (20) GSGSGRR, m GSGSGRR₅₇-b- 89,000 1.103 57 (60) 93,600 1.053 13~60 OEG₁₃ (20) GSGSGK, m ~8 GSGSGK₁₀-b- 9,650 1.047 10 (8)  17,900 1.04423 OEG₂₃ (20) GSGSGK, m ~15 GSGSGK₁₃-b- 12,100 1.022 13 (15) 17,800 1.1616 OEG₁₆ (20) GSGSGK, m ~30 GSGSGK₂₆-b- 24,800 1.056 26 (30) 30,7001.042 17 OEG₁₇ (20) GSGSGK, m ~60 GSGSGK₅₃-b- 50,800 1.114 53 (60)57,900 1.165 20 OEG₂₀ (20) GSGSGKK, m ~8 GSGSGKK₈-b- 7,170 n.d. 8 (8)14,900 n.d. 22 OEG₂₂ (20) GSGSGKK, m GSGSGKK₁₆-b- 13,700 n.d. 16 (15)19,800 n.d. 17 ~15 OEG₁₇ (20) GSGSGKK, m GSGSGKK₃₃-b- 29,000 n.d. 33(30) 35,600 n.d. 19 ~30 OEG₁₉ (20) GSGSGKK, m GSGSGKK₆₁-b- 54,000 n.d.61 (60) 64,500 n.d. 30 ~60 OEG₃₀ (20) KLA full length, KLA₆ 10,300 n.d.6 (5) — — — m ~5 KLA full length, KLA₁₁ 20,200 n.d. 11 (10) — — — m ~10KLA full length, KLA₁₄ 25,000 n.d. 14 (15) — — — m ~15 KLA fragment, mKLA(fragment)₁₁ 11,200 n.d. 11 (10) ~10 GSGSG-derived block copolymersand KLA homopolymers. ^(a)The name of the polymer as referred to in thetext. Note that the approximate degree of polymerization (DP) is givento best compare similarly sized polymers. Also note that the OEG coblockis omitted from the name. ^(b)The IUPAC name of the polymer with theactual DPs of blocks in the block copolymer. All polymers areend-labeled with a copy of fluorescein. ^(c)Number average molecularweight, except for GSGSGKK and KLA polymers, which are weight averagemolecular weight (M_(w)) ^(d)Dispersity of each block.^(e)Experimentally determined DPs with the theoretical DPs, based on theinitial monomer-to-initiator ratio, given in parentheses. These valueswere obtained by SEC-MALS except for those describing the GSGSGKK andKLA polymers, which did not elute on the SEC column. All values forthese polymers were determined by batch-mode static light scatteringwithout the SEC component. As such, no information on the dispersity ofthese polymers was obtained. The SEC-MALS chromatogram for all m ~60GSGSG derivatives that do elute on the SEC column were obtained. Allpeptide values are calculated with a dn/dc of 0.179. The OEG block has adn/dc of 0.11 and so a ratio (based on the initial monomer-to-OEG ratio)was used to calculate values for this block: the dn/dc from m ~8, 15, 30and 60 were 0.131, 0.141, 0.151 and 0.161, respectively.

In general, these data indicated that Arg residues appended to theC-terminus (GSGSGR, SEQ ID NO: 14 or GSGSGRR, SEQ ID NO: 15) exhibitedbetter intracellular penetration than the internally buried N-terminalderivatives (RGSGSG, SEQ ID NO: 12; RRGSGSG, SEQ ID NO: 13) (FIG. 19B).Peptides containing two Arg residues showed more robust fluorescentsignals when polymerized than those containing only one in the sameposition. Interestingly, in all cases, the Arg-containing peptidepolymers gave slightly lower values than that of a polymer prepared bypolymerizing a single Arg reside (R control polymer), which is themaximum theoretical signal that can result from a polymer containing oneArg per polymer side chain. In addition, peptides containing one or twolysine residues were taken up by cells when prepared as polymers (butnot as peptides alone), indicating that the presence of primary amino orguandinium units was sufficient for uptake. Moreover, the extent ofuptake of each polymer was shown to be dependent upon both the degree ofpolymerization (FIG. 25) and the concentration of material (FIG. 26),suggesting that uptake of these peptides can be improved by increasingeither factor.

Many bioactive peptides already contain one or more cationic amino acidsin their sequence. Therefore, it was demonstrated whether one suchpeptide could penetrate cells upon polymerization without the appendageof additional Arg or Lys residues. Moreover, it was determined if thepeptide maintain its intended biological function when incorporated intoa polymer in this manner (FIGS. 20A-20D). For this purpose, a knowntherapeutic peptide, KLA was chosen (sequence: KLAKLAKKLAKLAK), thatdoes not penetrate cells at sub-millimolar concentrations despite havingmultiple Lys residues in its parent sequence. KLA was shown to functionby lysing cellular mitochondria, resulting in apoptosis of the cell.However, because KLA does not inherently penetrate cells, to function itmust be conjugated to a CPP, prepared as a multimer, or appended to amolecular transporter.

Cell Penetration of KLA

To ascertain whether KLA could penetrate cells as a polymer brush, thepeptide was polymerized to varying DPs (DP or “m” in FIG. 20A areapprox. 5, 10 and 15). At each DP, the polymers gave strong fluorescencesignals by flow cytometry, similar to the Tat peptide control (FIG.20B), whereas the KLA peptide yielded fluorescence signalsindistinguishable from that of the vehicle control. Live-cell confocalmicroscopy verified internalization of the homopolymers at each Z-slicedepth. Live-cell confocal microscopy images were obtained showingaverage intensities from six consecutive 1 μm slices of HeLa cellstreated with the KLA peptide or polymer (m˜10).

Biological Function of the KLA Peptide

The reported biological function of the KLA peptide was determined,namely cytotoxicity by way of mitochondrial disruption, was not affectedby polymerization. Validating this notion, KLA polymers demonstrateddose-dependent cytotoxicity in HeLa cells (where concentration for allcytotoxicity studies is with respect to peptide) with LD₅₀ values in therange of what is seen for KLA-CPP conjugates (FIG. 20C). Furthermore, nocytotoxicity was detected for the unmodified KLA peptide, presumably dueto its inability to penetrate cells (FIGS. 19A-19B).

Cell Toxicity by Polymers not Caused by Polymer Scaffold

To confirm that the cell toxicity exhibited by the polymers was notcaused by the polymer scaffold or by internalization of any cationicpeptide polymer, performed the same assays with the GSGSG, GSGSGKK andGSGSGRR polymers (each at m˜60) were performed. No cytotoxicity wasexhibited by any of these materials at concentrations up to 1 mM. Inaddition, polymers composed of an analogue of the KLA sequence(KLA_(fragment)) with fewer Lys-Leu-Ala repeats (i.e., KLAKLAK, m˜10),in which the total number of amino acids is identical to that of thefull length KLA polymer at m˜5, exhibited negligible toxicity, despitehaving the ability to enter cells (FIG. 20B). This is likely because thesecondary structure of this peptide polymer, which is important for thetoxicity of KLA, differed dramatically from that of the KLA peptide andits direct polymer analogue. Importantly, these data clearly indicatethat the full-length KLA sequence was necessary for toxicity and notsimply a high density display of sequences with multiple lysines.Complimentary experiments confirmed that the cytotoxicity of the KLApolymers was the result of a disruption of mitochondrial membranepotentials (FIG. 20D and FIGS. 31A-31I) leading to mitochondiraldependent apoptosis of HeLa cells (FIG. 32), much like their KLA-CPP ormultimeric KLA analogues, further verifying that the key function of thepeptide is not perturbed by polymerization.

Route of Cellular Entry

The route of cellular entry was determined by employing thermal andpharmacological inhibitors of known uptake pathways. In all cases, theuptake of the materials was similarly affected by the inhibitors tested.These data, especially the results from dynasore, an inhibitor of thekey endocytosis player dynamin, indicate that polymers enter cells byendocytosis in a manner similar to the Tat peptide.

Resistance to Proteolytic Degradation of Peptides

Resistance of peptides to proteolytic degradation was confirmed.Analysis of reverse-phase HPLC (RP-HPLC) chromatograms before and afterproteolytic digestion indicated that while the peptide controls werecompletely degraded into fragments, the peptide polymers showed littleor no indication of proteolysis after incubation with multiple proteases(FIGS. 6A-6B).

In summary, a new method for rendering peptides cell penetrating byincorporating them into high density polymer brushes via graft-throughROMP was demonstrated. The only requirement for successful penetrationis the presence of a single Arg or Lys in the peptide sequence,preferably at the solvent-facing C-terminal end of the peptide. A knowntherapeutic peptide is the KLA peptide, which cannot enter cells on itsown, was shown to be cell penetrating by polymerization and,importantly, maintained its sequence-specific cytotoxic function as partof a polymer. This strategy offers the potential for the formulation ofa therapeutic with an exceptionally high weight percentage of the activepeptide (85% in the KLA homopolymer vs 50% for a Tat-KLA conjugate) thatis also resistant to proteolysis. Thus, a simple, effective and broadlyapplicable alternative to existing strategies that enable cellpenetration of peptides intended for medicinal or diagnostic purposes isdescribed in this disclosure.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A polymer having the formula: R¹-[M(O)]_(n)—R² wherein, n is aninteger from 2 to 1000; M is a polymerized monomer; O is independently atherapeutic polypeptide covalently attached to M through a covalentlinker; and R¹ and R² are independently terminal polymer moieties.
 2. Ablock copolymer having the formula:R¹-[M(O)]_(n)-[M(P)]_(m)—R² or R¹-[M(P)]_(m)-[M(O)]_(n)R² wherein, n isan integer from 2 to 1000; m is an integer from 2 to 1000; M is apolymerized monomer; O is independently a polypeptide covalentlyattached to M through a covalent linker; P is independently anon-polypeptide moiety; and R¹ and R² are independently terminal polymermoieties.
 3. A blend copolymer having the formula:R¹-([M(O)]_(n)-[M(P)]_(m))_(z)—R² or R¹-([M(P)]_(m)-[M(O)]_(n))_(z)—R²wherein, n is an integer from 2 to 1000; m is an integer from 2 to 1000;M is a polymerized monomer; O is independently a polypeptide covalentlyattached to M through a covalent linker; P is independently anon-polypeptide moiety; z is an integer from 2 to 100; and R¹ and R² areindependently terminal polymer moieties.
 4. The copolymer of claim 2,wherein said non-polypeptide moiety is a substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl and wherein said non-polypeptide moiety does not comprise anucleotide.
 5. The copolymer of claim 2, wherein said polypeptide is atherapeutic polypeptide.
 6. (canceled)
 7. The polymer of claim 1,wherein the polymerizable monomer isN-substituted-5-norbornene-2,3-dicarboximide, wherein the substitutioncomprises the therapeutic polypeptide.
 8. The polymer of claim 1,wherein M(O) is

wherein, L¹ is is independently a bond, —O—, —NH—, —COO—, —S—, —SO₂—,—SO₃—, —SO₄—, —SO₂NH—, —NHC(O)—, —C(O)NH—, —NHC(O)O—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and R⁴ is a polypeptide. 9.The polymer of claim 1, wherein the polypeptide comprises from 2 to 6arginine residues.
 10. The polymer of claim 1, wherein the polypeptidecomprises 2 arginine residues.
 11. The polymer of claim 9, wherein thearginine residues are the carboxyl terminal residues of the therapeuticpolypeptide.
 12. The polymer of claim 1, wherein the polypeptide doesnot comprise a carboxy terminal lysine residue.
 13. The polymer of claim1, wherein the polypeptide is resistant to proteolysis relative to theunpolymerized polypeptide.
 14. The polymer of claim 1, wherein R¹comprises a substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl.
 15. Thepolymer of claim 1, wherein R² comprises a substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl or substituted or unsubstitutedheteroaryl.
 16. The polymer of claim 1, wherein the polymer comprises adetectable moiety.
 17. A micelle comprising the copolymer of claim 2.18. The micelle of claim 17, having a longest dimension of between about1 and about 1000 nm. 19.-20. (canceled)
 21. A nanoparticle comprisingthe copolymer of claim
 2. 22. The nanoparticle of claim 21, wherein saidnanoparticle is a spherical nanoparticle.
 23. The nanoparticle of claim21, having a longest dimension of between about 1 and about 1000 nm.24.-25. (canceled)
 26. A method of administering a polypeptide to theinterior of a cell comprising contacting said cell with the polymer ofclaim
 1. 27. The method of claim 26, wherein said cell is in a subject.28. The method of claim 27, wherein said polymer is administeredsystemically to said subject.
 29. A method of treating a disease in asubject, comprising administering to said subject a polymer of claim 1.30. A pharmaceutical composition including a pharmaceutically acceptableexcipient and a polymer of claim
 1. 31. The copolymer claim 3, whereinsaid non-polypeptide moiety is a substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl andwherein said non-polypeptide moiety does not comprise a nucleotide. 32.The copolymer of claim 3, wherein said polypeptide is a therapeuticpolypeptide.
 33. A micelle comprising the copolymer of claim
 3. 34. Themicelle of claim 33, having a longest dimension of between about 1 andabout 1000 nm.
 35. A nanoparticle comprising the copolymer of claim 3.36. The nanoparticle of claim 35, wherein said nanoparticle is aspherical nanoparticle.
 37. The nanoparticle of claim 35, having alongest dimension of between about 1 and about 1000 nm.